Title page
Antiviral and immunoregulatory roles of Natural Killer cells in chronic hepatitis B virus infection
A THESIS PRESENTED TO UNIVERSITY COLLEGE LONDON
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY BY
DIMITRA PEPPA
APRIL 2013
DEPARTMENT OF IMMUNOLOGY AND MOLECULAR PATHOLOGY,
DIVISION OF INFECTION AND IMMUNITY,
UNIVERSITY COLLEGE LONDON
UNITED KINGDOM
1 Declaration
'I, Dimitra Peppa, confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis.'
2 Abstract
Persistent infection with hepatitis B virus (HBV) is a global health burden accounting for more than a 1 million deaths worldwide. Much work has focused on the failure of the adaptive immune response in persistent HBV infection. In contrast innate responses remain poorly characterised. Accumulating evidence indicates the involvement of natural killer (NK) cells in the control of viral infections and shaping of adaptive immunity. Here we investigated the antiviral and immunoregulatory potential of NK cells in chronic Hepatitis B virus infection (CHB). We found that the combination of immunosuppressive cytokines seen in CHB suppresses the non-cytolytic antiviral function of NK cells, whilst maintaining TRAIL mediated killing, which has been associated with hepatocyte apoptosis. Our findings also implicate NK cells in the down-regulation of antiviral T cells, which are profoundly diminished and prone to apoptosis in CHB. Our data show that in vitro depletion of NK cells results in recovery of HBV-specific CD8+ T cells, an effect that was not observed for control virus responses. When NK cells were depleted or not in contact with T cells, the degree of caspase activation in HBV-specific T cells was decreased, supporting a direct and contact dependent NK cell effect on virus-specific CD8+ T cell survival involving induction of apoptosis. NK cells, the predominant population of TRAIL expressing cells in the HBV infected liver, were found in intimate contact with T cells within the liver sinusoidal spaces.
The observed upregulation of the death receptor, TRAIL-R2 on CD8+ T cells, imposed by the intrahepatic milieu in HBV infection may sensitise them to apoptosis. Our findings illustrate an important and novel mechanism of immune dysregulation contributing to viral persistence in humans. The potential to selectively deviate NK cells from pathogenic roles, whilst augmenting antiviral functions could improve HBV control and enhance the design of future therapeutic strategies.
3 Acknowledgements
I am indebted to Ian Weller for his mentorship and for steering me in the direction of Mala Maini. Mala has been an excellent PhD supervisor. I am truly thankful for the confidence she has shown in me, for always being available for informal chats, for her many words of encouragement and for helping me to maintain a clear focus. Her drive and enthusiasm continues to inspire me. I would also like to thank my co-supervisor, John Trowsdale, for his valuable advice and help over the last four years.
None of this work would have been possible without the support from all the members, past and present, of the Maini group. Thank you all for creating such a great working environment, your technical advice, day-to-day help, unlimited supply of caffeine, bottles of wine and importantly your friendship.
I would also like to extend my gratitude to our collaborators, clinic staff and patients. In particular, Upkar Gill at the Royal London Hospital for his tireless ability to recruit patients for our study, Chiwen Chang at Cambridge for his help, and Gary Reynolds at Birmingham for his expertise in immunohistochemistry. A big thank you goes to Raymond Welsh and Stephen Waggoner for kindly agreeing to host my short visit at UMass, for providing me with a better insight of murine modeIs and for their thought provoking discussions. I am also grateful to the MRC for funding me during the duration of my PhD.
Finally I would like to thank my friends and family for always being there for me. My parents for teaching me to persevere in life, my sister Sofia and George for their unconditional love and support even from a distance and my little nephew, Nikolas, who always manages to put a smile on my face even when experiments are not working. Last but not least, thank you Nick for putting up with me, for counterbalancing Mediterranean emotions so effectively and for never failing to inject optimism and a sense of perspective into our lives. I couldn’t have managed through all the ups and downs without you.
4 List of common abbreviations
ADCC Antibody-dependent cellular cytotoxicity ALT Alanine aminotransferase APC Antigen presenting cell Bid Bcl2- interacting domain death agonist Bim Bcl2-interacting mediator Breg Regulatory B cell cccDNA Covalently closed circular DNA CCR7 Chemokine (C-C motif) receptor 7 CD Cluster of differentiation CD3z CD3zeta C-FLIP Cellular FLICE inhibitory protein CFSE Carboxyfluorescein diacetate succinimidyl ester CHB Chronic hepatitis B virus infection CMV Cytomegalovirus CpG Cytosine and guanine separated by phosphate CTL Cytotoxic T lymphocyte CTLA-4 Cytotoxic T-lymphocyte antigen 4 CXCR6 CXC-chemokine receptor 6 DAP-10 DNAX activating protein of 10kD DC Dendritic cell DD Death Domain DED Death effector domain DISC Death inducing signalling complex DNA Deoxyribonucleic acid EAE Experimental autoimmune encephalomyelitis E4BP4 E4 Promoter Binding Protein 4 EBV Epstein-Barr virus ELISA Enzyme-Linked ImmunoSorbent Assay EMCV Encephalomyocarditis virus ER Endoplasmic reticulum FACS Fluorescence activated cell sorter FBS Fetal bovine serum FADD Fas associated protein with death domain FLT3 Fms-like Tyrosine Kinase-3 GATA-3 GATA binding protein 3 GM-CSF Granulocyte-macrophage colony-stimulating factor HA Haemagglutinin HBcAg Hepatitis B virus core antigen HBeAg Hepatitis B virus precore-core antigen HBsAg Hepatitis B virus surface/envelope antigen HBV Hepatitis B virus HCC Hepatocellular carcinoma
5 HCV Hepatitis C virus HDV Hepatitis delta virus HIV Human immunodeficiency virus HLA Human leucocyte antigen HSC Hepatic stellate cell HPC Haematopoietic cell HTLV-1 Human T-cell Lymphotropic Virus ID-2 Inhibitor of DNA binding 2 iDC Immature dendritic cell IFN-α Interferon alpha IFN-γ Interferon gamma IFN-λ Interferon-lamda Ig Immunoglobulin ILC Innate lymphoid cell IL-10 Interleukin-10 IL-12 Interleukin-12 IL-15 Interleukin-15 IL-17 Interleukin-17 IL-18 Interleukin-18 IL-2 Interleukin-2 IL-22 Interleukin-22 IL-29 Interleukin-29 IL-6 Interleukin-6 IL-7 Interleukin-7 IL-8 Interleukin-8 INF-A Influenza A iNK Immature Natural Killer ITAM Immunoreceptor tyrosine–based activation motif ITIM Immunoreceptor tyrosine-based inhibitory motif JAK Janus kinase KC Kupffer cell KIR Killer immunoglobulin receptor KLRG-1 Killer cell lectin-like receptor subfamily G, member 1 LCMV Lymphocytic Choriomeningitis Virus LPS Lipopolysaccharide LSEC Liver sinusoidal epithelial cell LTA Lipoteichoic acid LTi Lymphoid tissue inducer MAPK Mitogen activated protein kinase MCMV Mouse cytomegalovirus MDSC Myeloid-derived dendritic cell MFI Mean fluorescence intensity MHC Major histocompatability complex MHV Murine hepatitis virus
6 MIP Macrophage inflammatory protein MTP Microsomal triglyceride transfer protein NCAM Neural cell adhesion molecule NCR Natural cytotoxicity receptor NEMO NF-kappa-B essential modulator NK Natural killer NKG2A Natural-killer group 2, member A NKG2C Natural-killer group 2, member C NKG2D Natural-killer group 2, member D NKp30 Natural killer cell p30-related protein NKp44 Natural killer cell p44-related protein NKp46 Natural killer cell p46-related protein NKP Natural killer precursor NK-R Natural killer receptor NKT Natural killer T NPC Non-parenchymal cells OPG Osteoprotegerin ORF Open reading frame PAMP Pathogen-associated molecular pattern PBMC Peripheral blood mononuclear cells PBS Phosphate buffered saline pDC Plasmacytoid dendritic cell PCR Polymerase chain reaction PD-1 Programmed death-1 Peg-IFN-α Pegylated-Interferon-α PI3K Phosphoinositide 3-kinase PLC-γ Phospholipase C gamma PMA Phorbol 12-Myristate 13-Acetate PMN Polymorphonuclear PRR Pattern recognition receptor RIP1 Receptor Interacting protein 1 RNA Ribonucleic acid RORγt Retinoic acid receptor related orphan receptor gamma SHP-1 Src homology 2 domain-containing phosphatase-1 SIV Simian immunodeficiency virus SLT Secondary lymphoid tissue SNP Single nucleotide polymorphism STAT-1 Signal transducer and activator of transcription 1 Tcm Central memory CD8+ T cells TCR T cell receptor Tem Effector memory CD8+ T cells Temra Revertant CD8+ T cells TGF-β Transforming growth factor beta Tim-3 T cell immunoglobulin mucin-3
7 TLR Toll-like receptor TNF-α Tumour necrosis factor alpha TNFR1 Tumour necrosis factor receptor 1 TRADD TNF-receptor associated death domain TRAF-2 TNF-receptor associated protein TRAIL TNF-related apoptosis inducing ligand TRAIL-R TNF-related apoptosis inducing ligand receptor Treg Natural regulatory T cell uPa/SCID Urokinase-type plasminogen activator-severe combined immunodeficiency VV Vaccinia virus WHV Woodchuck hepatitis virus ZAP-70 Zeta-chain-associated protein kinase 70
8 Table of Contents
Title page ...... 1 Declaration ...... 2 Abstract ...... 3 Acknowledgements ...... 4 List of common abbreviations ...... 5 List of Figures ...... 12 List of Tables ...... 13 Thesis Outline ...... 14 1. Introduction ...... 15 Public health significance of Hepatitis B virus Infection and limitations of current treatment ...... 15 HBV replication - Unique features of the virus ...... 17 Natural history and clinical course of HBV ...... 22 Immune responses against HBV- Host Factors ...... 24 Innate immune response ...... 25 Adaptive immune response ...... 29 Immunopathology of CHB and NK cells – Multiple hats for NK cells? ...... 34 NK cells general aspects ...... 39 NK cell developmental origins and subsets ...... 40 NK cell receptors and education ...... 45 NK cell arsenal of weapons and regulation - an overview ...... 51 NK cells in antiviral defense – viral recognition and genetic studies ...... 55 NK cell cellular crosstalk and immunoregulatory role ...... 62 Emerging concept in NK cell biology. Do NK cells remember? ...... 66 NK cells in context: the liver microenvironment ...... 68 NK cells in HBV infection ...... 73 Aims of the study ...... 73
2. Material and Methods ...... 74 Antiviral role ...... 74 Separation of PBMC ...... 74 Media ...... 75 Processing of liver samples and isolation of intrahepatic lymphocytes ...... 75 Antibodies and reagents for analysis of NK cells ...... 75
9 Extracellular staining and Flow Cytometric analysis ...... 77 IFN-γ production by intracellular staining ...... 77 CD107 degranulation assay ...... 77 Determination of Serum Cytokine concentrations by Cytometric Bead Array (CBA) ...... 78
Regulatory role ...... 78 NK cell isolation and sorting ...... 78 Antibodies and reagents ...... 79 Identification of virus specific CD8+ T cells ...... 82 Peptide stimulation ...... 82 Short-term culture ...... 83 Overnight stimulation ...... 84 Immunohistochemistry and Immunofluorescence ...... 85 Statistical analysis ...... 85
3. Antiviral NK cell role in chronic HBV infection ...... 87 Background ...... 87 Study Cohort ...... 91 Results ...... 93 3.1 Expansion of CD56bright subset ...... 93 3.2 Impaired non-cytolytic antiviral potential of NK cells in CHB ...... 96 3.3 IL-10 is induced in CHB and recapitulates the NK cell defect in IFN-γ production ...... 103 3.4 Restoration of NK cell IFN-γ production upon blockade of immunosuppressive cytokines ...... 108 Discussion ...... 115
4. Regulatory NK cell role and involvement of death receptors in the modulation of T cell responses in CHB ...... 123 Background ...... 123 Patients and healthy controls ...... 131 Results ...... 137 4.1 Depletion of NK cells rescues virus specific CD8+ T cells ...... 137 4.2 Differential effects of NK cells according to T cell specificity ...... 140 4.3 NK cells limit HBV-specific CD8+ responses in a contact dependent manner by inducing apoptosis ...... 143 4.4 Increased TRAIL-R2 expression on T cells in CHB ...... 147 4.5 Further upregulation of TRAIL-R2 on intrahepatic CD8+ T cells in CHB ...... 152
10 4.6 TRAIL-R2 expression: a hallmark of T cells encountering their cognate antigen in the HBV-infected liver ...... 156 4.7 TRAIL blocking partially recovers HBV-specific CD8+ T cells ...... 160 4.8 Overnight rescue of intrahepatic HBV-specific T cells by TRAIL blockade ...... 163 Discussion ...... 167
5. Ongoing work & Future Directions ...... 176 Summary of work presented and unanswered questions ...... 176 Are CD4+ T cells amenable to NK mediated deletion in CHB infection and is the TRAIL pathway involved? ...... 179 Additional pathways mediating deletion ...... 185 Future Outlook: Explore the role of NK cells in antiviral immunity in other chronic viral infections ...... 190
List of publications and abstracts ...... 194 References ...... 197
11 List of Figures
Figure 1A. HBV genome...... 18 Figure 1B. HBV Replication Cycle...... 21 Figure 1C. Clinical course of a) acute and b) chronic HBV infection...... 22 Figure 1D. Schematic representation of molecular defects...... 31 Figure 1E. Potential receptor/ligand interactions in the liver and potential mechanisms of injury ...... 38 Figure 1F. Model of human NK cell development ...... 45 Figure 1G. Interactions with adaptive and innate immune cells...... 65 Figure 1H. Effector properties and NK cell interactions within the hepatic sinusoid during steady state (A) and viral infection (B) ...... 72 Figure 2A. NK cell purity...... 78 Figure 2B. Representative example of gating strategy...... 82 Figure 3.1 NK cell frequency and altered subset distribution in the periphery and intrahepatic compartment ...... 95 Figure 3.2 Higher TRAIL expression and reduced IFN-γ production by NK cells in CHB ...... 98 Figure 3.2.1 Defective IFN-γ production by both NK subsets in CHB ...... 100 Figure 3.2.2 Skewed NK cell effector function in CHB is only partially corrected during therapy ...... 102 Figure 3.3 Higher levels of circulating IL-10 in CHB ...... 105 Figure 3.3.1 Effect of exogenous IL-10 ...... 107 Figure 3.4 IL-10 blockade alone or in combination with TGF-βRII blocking restores NK cell IFN-γ production ...... 109 Figure 3.4.1 Trend towards lower IFN-γ production by the CD56bright NK cell subset in the HBV infected liver ...... 112 Figure 3.4.2 Blockade of IL-10/TGF-β enhances intrahepatic NK cell IFN-γ production ...... 114 Figure 4A. The extrinsic and intrinsic pathways of apoptosis...... 126 Figure 4B. TRAIL receptors and signaling...... 128 Figure 4.1 Recovery of HBV-specific CD8+ T cells following depletion of NK cells ...... 138 Figure 4.2 Differential regulation by NK cells according to T cell specificity ...... 141 Figure 4.3 NK cells limit the survival of CD8+ HBV-specific T cells in a contact dependent manner by inducing apoptosis ...... 146 Figure 4.4 Higher levels of TRAIL-R2 on T cells in patients with CHB ...... 151
12 Figure 4.5 Intrahepatic CD8+ T cells in CHB patients have upregulated expression of TRAIL-R2 ...... 155 Figure 4.6 TRAIL-R2 expression is a feature of CD8+ T cells encountering antigen in the HBV- infected liver ...... 159 Figure 4.7 Partial recovery HBV-specific CD8+ T cells to TRAIL blockade ...... 162 Figure 4.8 Overnight recovery of intrahepatic HBV-specific T cells by TRAIL blockade ...... 166 Figure 5.1 Working hypothesis of the antiviral and regulatory role of NK cells in CHB...... 178 Figure 5.2 Ex vivo killing capacity of NK cells ...... 181 Figure 5.3 Increased proportions of activated HBV-specific CD4+ T cells in the absence of NK cells ...... 182 Figure 5.4 Increased TRAIL-R2 expression on CD4+ T cells in CHB patients ...... 183 Figure 5.5 Limited recovery of intrahepatic HBV-specific T cells by TRAIL blockade ...... 184 Figure 5.6 Conserved expression of NKG2D on NK cells from CHB patients ...... 187 Figure 5.7 Depiction of 2B4 model system used...... 188 Figure 5.8 NKG2DL expression on PBMCs from CHB patients and healthy controls based on GFP expression on 2B4 cell line...... 189
List of Tables
Table 1.1 Illustration of the NK cell interaction repertoire in humans...... 46 Table 1.2. Evidence for antiviral role of NK cells ...... 60 Table 3.1. Characteristics of study population ...... 91 Table 3.2. Patient characteristics with available liver biopsy specimens ...... 92 Table 4.1. Characteristics of patients from whom PBMC alone were available...... 132 Table 4.2. Characteristics of CHB patients with available paired PBMC and liver biopsy specimens...... 133 Table 4.3. Patient characteristics with available liver biopsy specimens used as controls. .. 135 Table 4.4. HCV patient characteristics with available PBMC...... 136
13 Thesis Outline
Chapter 1, (Introduction), outlines the epidemiology, public health significance, natural course of HBV and key virus and host factors in the pathogenesis of chronic HBV infection
(CHB). Sections on innate and adaptive immunity during the course of HBV infection, detailing the shortcomings of the immune response to control the virus, are discussed. This chapter draws on a previously published review (Peppa and Maini 2012) (Maini and Peppa
2013). A broad overview of Natural Killer (NK) cell biology and their role against viral infections is provided, with more focussed sections describing them in the context of the liver environment and potential dual roles in chronic HBV infection.
Material and methods, outlined in Chapter 2, include comprehensive details of protocols and reagents used for the undertaking of this work.
In Chapter 3, we evaluated the phenotypic and functional characteristics of NK cells from the periphery and liver of chronically infected HBV patients, and demonstrate an NK cell dysfunction in IFN-γ production. The potential mechanisms contributing to this defect in non-cytolytic effector function with emphasis on the effect of the cytokine environment and the action of immunosuppressive cytokines are described (Peppa, Micco et al. 2010). On the basis of this functional defect identified, we hypothesised that in CHB, NK cells are deviated from an antiviral towards an immunoregulatory role. Of note the combination of immunosuppressive cytokines seen in CHB was found to suppress the non-cytolytic antiviral function of NK cells, whilst maintaining TRAIL mediated killing. These data formulated the basis for investigating the regulatory potential of NK cells, in terms of their ability to impair T cell mediated control of HBV by deleting virus specific T cells (Chapter 4) (Peppa, Gill et al.
2013). Chapters 3 and 4 have been previously published and therefore the results are described concisely in this thesis. The introduction and discussion sections are however more thoroughly presented.
In Chapter 5, we summarise our findings and explore future directions.
14 1. Introduction
The immune mechanisms that favour elimination of hepatocellular antigens and/or contribute to liver immunopathology in hepatitis B virus (HBV) infection are not fully elucidated. The HBV-specific CD8+ T cells constitute a critical component of antiviral defence, which are profoundly debilitated during persistent infection. However, the adaptive and innate arm of the immune response rarely act in isolation, and the interplay between these two sides of the immune system contributes to the most productive overall response and viral clearance. Although recent advances have increased our understanding and ability to carefully dissect the capacity of innate components of the immune response to induce both immunopathology and resolution of HBV infection, their involvement is just beginning to be appreciated. Natural Killer (NK) cells are one of the major effectors of innate immunity that may have divergent effects during HBV infection. The study of NK cells in chronic HBV infection (CHB) could lead to the identification of specific components that can be successfully exploited to control viraemia and/or moderate excessive liver damage. This work may also generate exciting new insights into the reciprocal regulation between adaptive and NK cell mediated innate immunity, which may be applicable to other chronic viral infections.
Public health significance of Hepatitis B virus Infection and limitations of current treatment
Infection with HBV and its chronic sequelae account for approximately one million annual deaths, remaining a major and challenging global public health problem. Even though a prophylactic HBV vaccine (Lavanchy 2004) is available, WHO estimates that two billion people have been infected with HBV, and more than 350 million have persistent infection.
Liver inflammation and progressive fibrosis are the hallmarks of continuous liver injury
15 extending over time to cirrhosis, liver failure and hepatocellular carcinoma (HCC), the major complications of HBV (Liaw and Chu 2009). The incidence of HBV-related HCC is estimated to rise in the next two decades given the high numbers of chronic HBV carriers and the protracted course to the development of HCC (Nguyen, Law et al. 2009). The carrier frequency varies depending on the geographical area from low (0.1-2%) in USA and western
Europe, to intermediate (2-8 %) in Mediterranean countries, and high (8-20%) in sub-
Saharan Africa and parts of Asia (Lavanchy 2004) (Liaw and Chu 2009). In areas of high endemicity, the vast majority of individuals acquire infection either vertically or in early childhood, whereas in the low prevalence areas, the infection is acquired primarily in adulthood (Lavanchy 2004). Although, there is a reported low prevalence of CHB in the UK, in recent years the number of infected individuals has doubled, mainly due to globalisation and changing migration patterns from countries of intermediate or high HBV prevalence
(Hepatitis B Foundation-2008). Despite this, a universal vaccination programme remains to be implemented in the UK.
A complex interaction between virological and host factors appears to be involved in HBV persistence, circumventing the activation of the host’s protective antiviral responses. The large pool of chronically infected patients requires antiviral treatment, which can be a lifelong commitment, associated with high cost and the additional problems of emergence of viral resistance and drug toxicity. Although, the availability of potent antiviral drugs such as Entecavir or Tenofovir with a higher genetic barrier to resistance has improved the treatment of CHB, eradication or long-lasting immune responses are still rarely achieved
(Papatheodoridis, Manolakopoulos et al. 2008). Treatment with pegylated Interferon-α
(Peg-IFN-α) has the advantages of a finite duration of treatment, the absence of resistance and immunomodulating effects potentiating durable virological responses. Yet only a minority of patients respond to IFN-α therapy, which is often limited by low tolerability and
16 toxicity (Papatheodoridis, Manolakopoulos et al. 2008). Interestingly in our recent study, treatment with Peg-IFN-α mediated functional augmentation of NK cell effector function, which correlated with peak virological responses, highlighting the antiviral potential of NK cells (Micco, Peppa et al. 2012). Thus a comprehensive analysis of both arms of the immune response during HBV infection may lead to new adjuvant immunotherapeutic approaches and strategies to restore the defective anti-viral immunity present in patients with chronic infection and reduce disease burden.
HBV replication - Unique features of the virus
HBV has several key features implicated in the pathogenesis and clinical outcome of HBV infection. Classified as a hepatotropic virus belonging to the Hepadnaviridae family, it replicates in the liver, where almost all hepatocytes could succumb to HBV infection. The virus itself is not directly cytopathic and liver injury is thought to be the result of immune mediated attempts to control the infection (Ando, Moriyama et al. 1993; Guidotti, Rochford et al. 1999; Thimme, Wieland et al. 2003) (Bertoletti, Maini et al. 2010). HBV is a partially double stranded DNA virus, whose genome encodes four overlapping reading frames (ORFs); the preS/S encoding the three viral surface proteins; the precore/core encoding the core protein, a structural unit of the viral capsid, and the non structural pre-core protein, the secreted e-Antigen (HbeAg); the pol ORF encoding the viral polymerase required for RNA encapsidation and DNA synthesis; and the X ORF encoding the small viral regulatory X protein (HBx), which is essential for viral replication (Dandri and Locarnini 2012) (Fig. 1A).
Different size envelope proteins, small (S), medium (M) and large (L) share the same C- terminal domain, which contains the hepatitis B surface antigen (HBsAg) (Ganem and
Varmus 1987).
17
Figure 1A. HBV genome. The inner circles represent the full-length minus (-) strand (with the terminal protein attached to its 5' end) and the incomplete plus (+) strand of the HBV genome. The outermost coloured lines indicate the translated HBV proteins: large, middle and small HBV surface proteins, polymerase protein, X protein, and core and pre-core proteins. Modified from Rehermann & Nascimbeni 2005.
Essential steps of HBV viral replication, such as viral entry remain poorly understood, despite years of intensive research. It had been proposed that following viral attachement to liver cell associated heparan sulphate proteoglycans, the virus binds to a hepatocyte specific pre-
S1 receptor. Along these lines, an N-terminal myristoylated peptide corresponding to the pre-S1 domain amino acids 2-48 of the L protein, has been effective in preventing both HBV and hepatitis delta virus (HDV) infections of hepatocytes through most likely the engagement of a viral receptor (Barrera, Guerra et al. 2005) (Gripon, Cannie et al. 2005)
(Schulze, Schieck et al. 2010). In a more recent study a transmembrane transporter predominately expressed in the liver, sodium taurocholate cotransporting polypeptide
(NTCP), has been identified to interact with the L proteins of HBV and HDV in a specific
18 manner functioning as the potentially common and so far elusive viral receptor (Yan, Zhong et al. 2012). Following binding of the virus the nucleocapsid is released into the cytoplasm.
The HBV genome is then transferred to the nucleus, where the partially double-stranded
DNA genome is converted into a covalently closed circular DNA (cccDNA) minichromosome, establishing a lifelong HBV reservoir (Locarnini and Zoulim 2010). The persistence of this transcriptional template in hepatocytes can lead to HBV reactivation in some inactive carriers (Fattovich, Olivari et al. 2008). One of the main challenging tasks of antiviral treatments is to completely eliminate or restrict the formation of cccDNA(Nassal 2008).
Following the establishment of cccDNA, viral messenger RNAs are transcribed, including pre- genomic RNA (pgRNA), the template for reverse transcription of viral DNA. Viral RNAs are transported to the cytoplasm, where they are used as mRNAs for the translation of HBV surface, core, polymerase and X proteins. Nucleocapsids are then assembled in the cytoplasm, into which a strand of pgRNA is encapsidated with the polymerase protein, and is reverse transcribed inside the nucleocapsid to minus-strand DNA, which is then used to synthesize plus-strand DNA by DNA polymerase (Ganem and Varmus 1987; Rehermann and
Nascimbeni 2005) (Urban, Schulze et al. 2010). The DNA-containing capsid migrates biderectionally within the cytoplasm. One pathway terminates at the endoplasmic reticulum
(ER) membrane, where it interacts with the envelope proteins triggering an internal budding reaction, resulting in the formation and secretion of virions. The second pathway transports the maturing capsid back to the nucleus, where the cccDNA pool can be further amplified
(Ganem and Varmus 1987; Urban, Schulze et al. 2010) (Fig. 1B). Contrary to all known mammalian DNA viruses, HBV replicates through reverse transcription of an RNA intermediate. The lack of proof-reading activity of the viral polymerase accounts for the emergence of genetic heterogeneity of HBV and viral populations containing mutations affecting the production of e-Antigen (eAg), as well as mutations confering resistance to
19 antiviral therapy (Locarnini and Zoulim 2010). Particular selection pressures, arising from host immune clearance (endogenous) as well as exogenous pressures secondary to the use of antivirals and vaccines, can therefore select out new escape mutants. Ten HBV genotypes have been described in total with a distinctive geographical prevalence. In the recent years there is growing evidence that HBV genotypes and subgenotypes influnce clinical outcomes and may play a role in the host-virus relationship (Tanwar and Dusheiko 2012).
Another unique feature of HBV is the capacity of an infected cell to secrete large amounts of circulating HBeAg and 20nm sphere and filamentous HBsAg particles, which do not contain the HBV genome and outnumber virions by a factor of 104-106 (Ganem and Prince 2004)
(Rehermann and Nascimbeni 2005). These antigens are postulated to have an immunomodulatory function that protects the virus against immune attack (Wieland and
Chisari 2005) (Milich and Liang 2003). HBeAg, the secretory form of the core antigen, which is not required for viral replication, has been postulated to induce tolerance in utero, predisposing neonates born to HBV-infected mothers to chronic infection (Milich, Jones et al. 1990). Although, the production of HBeAg and HBsAg has been associated with hampering of adaptive immune mechanisms and T cell exhaustion (Reignat, Webster et al.
2002; Milich and Liang 2003; Chen, Sallberg et al. 2005), a careful evaluation of their effect on innate immunity is lacking. Some studies have suggested a possible suppression of toll- like-receptor (TLR) mediated innate immune responses by HBV secretory proteins (Wu,
Meng et al. 2009) (Visvanathan, Skinner et al. 2007). Moreover, the HBx protein has the potential when overexpressed to inhibit the processing and presentation of viral antigen
(Wieland and Chisari 2005). Overall, these key viral factors may favour persistence and perpetuate tolerance of the various effector arms of the immune response.
20
Figure 1B. HBV Replication Cycle. Adapted from (Zoulim and Locarnini 2009).
21 Natural history and clinical course of HBV
Acute HBV infection is characterised by a long delay prior to an exponential increase in HBV viral load. HBV DNA increases and declines prior to the peak symptoms of acute hepatitis
(increase in the levels of alanine aminotransferase (ALT)) development of jaundice and induction of a cell mediated immunity (Webster, Reignat et al. 2000) (Guidotti, Ishikawa et al. 1996) (Chang and Lewin 2007) (Dunn, Peppa et al. 2009) (Fig. 1C). The age at infection primarily determines the rate of progression from acute to chronic infection. 1 to 5% of infected adults, 20 to 30% of young children and up to 90% of perinatally infected individuals develop persistent infection with HBV (Liaw and Chu 2009). Resolution, characterised by
HBsAg loss from the blood within 6 months of infection, generally results in lifelong immunity. However, it is now increasingly recognised that HBV is contained by an effective immune response rather than being completely eradicated (Rehermann, Ferrari et al. 1996), with the potential to reactivate in the context of significant immunosuppression, such as co- infection with human immunodeficiency virus (HIV) or following bone marrow transplantation.
Figure 1C. Clinical course of a) acute and b) chronic HBV infection. Fluctuations of serological markers, serum HBV DNA and liver inflammation (ALT) over the typical course of acute and chronic HBV infection. Modified from Chang and Lewin 2007.
22 CHB follows a highly dynamic natural course that varies amongst infected individuals. To aid clinical categorisation of patients a combination of virological, serological and biochemical parameters have been traditionally used to describe four distinct phases: immune-tolerant; immune-clearance or immune-reactive; immune control/inactive (low or non-replicative); and HBeAg negative CHB (immune escape) (Villa, Fattovich et al. 2011) (Dandri and Locarnini
2012) (Fig. 1C). It is important to note that these phases do not always occur in sequence and in all patients with CHB. The immune-tolerant phase is characterised by high levels of
HBV DNA, serum HBeAg positivity, and a normal or minimally elevated ALT; perinatally infected children are predominantly considered to be in the immune-tolerant phase.
Following perinatal infection this phase is long-lasting, in contrast to those who acquire infection in adulthood. During the immune-clearance phase disease activity fluctuates and progressive liver damage occurs. Eventual seroconversion from HBeAg positive to anti-e antibody (anti-HBeAb) (20 to 30% per year) is usually followed by a non-replicative or inactive phase associated with low/undetectable HBV DNA and biochemical improvement of inflammatory activity (Chen, Yang et al. 2009). Occassionaly individuals will also clear HBsAg, but this occurs in less than 1% of patients per annum (Villa, Fattovich et al. 2011) (Fattovich,
Olivari et al. 2008). A subset of patients can develop HBV reactivation with either the wild type and reversion to HBeAg positivity (1-4%), or with HBV variants limiting eAg production
(Fattovich, Olivari et al. 2008), (Yuen, Sablon et al. 2002). Individuals with HBeAg negative
CHB, who now constitute the bulk of patients attending hepatology clinics in Europe, are susceptible to wide fluctuations in both viral replication and serum ALT and are at increased risk of accelerated disease progression (Brunetto, Giarin et al. 1991). Moreover, it should be emphasised that while these distinct phases may be useful for clinical categorisation, this nomenclature is not reflective of the immunological responses encountered during these phases. For instance, there is little evidence to support that the immune response is more tolerant in the immune-tolerant than the immune-reactive phase and typically T cell
23 responses have been found to be most vigorous in the inactive phase (Webster, Reignat et al. 2004) (Maini, Boni et al. 2000). A recent study of children and young adults with CHB in the immune-tolerant phase has further highlighted that ALT levels are a poor reflection of the presence or absence of an antiviral T cell response (Kennedy, Sandalova et al. 2012). The
T cell profile of immune-tolerant young patients was found to be similar to their immune- reactive counterparts and less compromised than that observed in older patients (Kennedy,
Sandalova et al. 2012). These data challenge the dogma that younger patients lack an HBV- specific adaptive immune response and therefore, suggest that young CHB patients are more suitable treatment candidates than previously considered (Ganem and Prince 2004)
(Kennedy, Sandalova et al. 2012).
The extent of chronic liver disease is linked to the frequency, duration and severity of necroinflammatory hepatic flares in patients in the HBeAg-positive or negative phases.
Hepatic flares can be complicated by hepatic decompensation in 2-3% of patients and characteristically these adverse events drive fibrosis leading to cirrhosis. The lifetime risk of developing cirrhosis in patients with CHB is 15 to 40% (Liaw and Chu 2009). CHB remains the main risk factor for the development of hepatocellular carcinoma (HCC), accounting for 50%
HCC cases worldwide and 70-80% of HCC cases in high endemic regions (Nguyen, Law et al.
2009). The multifactorial mechanisms which lead to the great burden of chronic infection and its complications (cirrhosis, hepatic decompensation and HCC) are not completely defined.
Immune responses against HBV- Host Factors
The majority of individuals exposed to HBV as adults are able to control acute infection, exemplifying the integral capacity of the immune system to successfully combat HBV
24 infection. The potential for immune control in the setting of established chronic infection has been further underscored by resolution of CHB in recipients following bone marrow transplantation from an immune donor (Ilan, Nagler et al. 1993) (Hui, Lie et al. 2005). The limited host range of HBV to humans and chimpazees, along with the drawbacks of current experimental models have posed a significant challenge in exploring the various immunological events during infection.
Innate immune response
The liver is an organ with predominant innate features due to the abundance of Kupffer cells
(KC), NK cells, Natural Killer T (NKT) cells, parenchymal and non parenchymal cells (NPCs) that can potentially sense and signal the presence of HBV, in addition to exerting direct antiviral effects and producing cytokines and chemokines that allow the recruitment and maturation of adaptive responses (Protzer, Maini et al. 2012). In the early antiviral state production of type I interferons (IFNs) is a major mechanism in response to infection (Akira,
Uematsu et al. 2006). Analysis of the intrahepatic gene expression in a seminal study from the chimpanzee model has, however, shown a lack of induction of type I IFN genes in the liver during the entry and expansion phase of HBV (Wieland, Thimme et al. 2004). These findings are supported by human studies, where the circulating levels of type I IFNs were scarcely detectable during the early course of infection (Dunn, Peppa et al. 2009) (Stacey,
Norris et al. 2009). From a clinical viewpoint and in marked contrast with other infections such as human immunodeficiency virus (HIV), human cytomegalovirus (HCMV) or dengue virus, HBV is characterised by delayed propagation and absence of ‘flu-like’ symptoms suggestive of an acute viral infection, supporting the case for a defective type I IFN production (Bertoletti, Maini et al. 2010). Consequently, HBV has been frequently referred to in the literature as a ‘stealth’ virus that can evade the radar of innate immunity (Wieland,
Thimme et al. 2004).
25 This concept has been, however, challenged by emerging lines of evidence arguing that HBV can elicit and then disarm innate immune responses. For instance it has been reported that high level HBV replication is capable of eliciting type I IFNs, but this response may be actively counteracted by liver non-parenchymal cells (NPC) with the ability to suppress it in vivo
(Bertoletti, Maini et al. 2010) (Durantel and Zoulim 2009). HBV replication in HepaRG cells, an overexpression system based on recombinant baculovirus that achieves high intracellular
HBV replication levels, induced IFN-I-stimulated genes, resulting in non-cytopathic suppression of HBV replication in the absence of NPCs (Lucifora, Durantel et al. 2010).
Infection with high dose viral inocula of woodchuck hepatitis virus (WHV) has also been found to induce intrahepatic gene expression (Guy, Mulrooney-Cousins et al. 2008). The physiological relevance of these data needs to be interpreted with caution in view of the artificial conditions of overexpressing HBV constructs in HepaRG cells and the high dose of
WHV utilised to establish infection in animals. Nonetheless these are important parameters that need to be taken into consideration given that both the quantity of DNA and size of infecting viral inoculum may influence the outcome of infection and triggering of innate host immunity (Asabe, Wieland et al. 2009) (Unterholzner and Bowie 2011). The lack of a robust infection system based on physiologically pertinent cells has been a major limiting factor in demonstrating the initial host-virus interactions. Interestingly, supporting the ability of the host’s innate immunity to detect HBV, a transient activation of IFN-α genes has been identified in human hepatocytes following HBV infection in chimeric mice (Lutgehetmann,
Bornscheuer et al. 2011).
It remains undefined how the innate immune system is capable of sensing HBV, camouflaged as a minichromosome (cccDNA) in the nucleus of an infected hepatocyte. Even though active HBV replication is not supported in non-parenchymal liver cells (Untergasser,
Zedler et al. 2006) and important questions remain regarding the detection of HBV early
26 after the onset of infection, alteration of innate immune responses by modulation of Toll- like receptor (TLR) expression and signalling have been reported. Evidence from Wu et al. suggested that HBV targets and disables the TLR system, correlating with attenuated IFN-β production and subsequent lack of induction of IFN-stimulated genes (Wu, Meng et al.
2009), supporting the concept that HBV has evolved strategies to actively sabotage the IFN pathways. In addition to HBV secretory proteins abrogating TLR-mediated signalling (Wu,
Meng et al. 2009), a role for HBeAg in downregulating functional TLR-2 expression in PBMCs,
KC and hepatocytes from HBV infected patients has also been suggested (Visvanathan,
Skinner et al. 2007), which may in turn contribute to the establishment of chronic infection.
Interestingly, TLR activation of the weak innate response to HBV is in fact showing promising results suppressing HBV replication in vitro and in transgenic mice, and TLR agonists are showing some efficacy in chimpanzees and woodchucks (Bertoletti and Ferrari 2012)
(Isogawa, Robek et al. 2005).
Recent observations suggest that HBV may employ multiple strategies to evade both TLR- dependent and independent siganlling pathways. Work from human-liver chimeric mice and in vitro models indicated that HBV might affect signal transducer and activator of transcription (STAT) methylation and IFN-α signalling (Lutgehetmann, Bornscheuer et al.
2011) (Christen, Duong et al. 2007) (Dandri and Locarnini 2012). Studies in hepatoma cell lines further suggested that HBV polymerase and X protein may inhibit downstream signalling of cytoplasmic DNA receptors, thereby preventing activation of the type-I interferon genes (Wang, Li et al. 2010; Kumar, Jung et al. 2011; Bertoletti and Ferrari 2012).
A remaining possibility is that type-III IFNs, which are preferentially produced in the liver and have been shown to have anti-HBV activity in the HBV transgenic mouse, may play a local rather than a systemic role, complementing or partially replacing the early antiviral role of
27 IFN-I (Robek, Boyd et al. 2005) (Bertoletti, Maini et al. 2010). Their role has been demonstrated in HCV infection, where a strong association exists between polymorphisms upstream of IFN-lamda 3 region and HCV clearance (Thomas, Thio et al. 2009) (Ge, Fellay et al. 2009) (Rauch, Kutalik et al. 2010). Evidence linking coding changes in the type I IFN receptor gene and a gene encoding a receptor chain for the IFN-lamda with HBV clearance
(Frodsham, Zhang et al. 2006), also highlights the potential valuable contribution of these
IFNs in the early control of HBV.
Alternatively, HBV may have evolved additional mechanisms to selectively modulate other early innate antiviral responses such as production of Interleukin-6 (IL-6); a cytokine recently shown to have the capability to constrain HBV replication. Transient IL-6 production by
Kupffer cells in response to HBV-infected primary human hepatocytes raised the possibility that the virus may be actively suppressing the action of IL-6, as part of the viral arsenal of immune evasion stategies (Hosel, Quasdorff et al. 2009). Moreover, the study of a cohort of patients in the early pre-clinical phase of HBV followed up until resolution of infection has drawn attention to the role of the immunosuppressive cytokine interleukin-10 (IL-10) in actively attenuating early innate (NK cell) effector responses (Dunn, Peppa et al. 2009). Thus production of IL-10 may serve as a window of opportunity for viral escape.
Although HBV transgenic mouse models have demonstrated that NKT and NK cells have the potential to contribute to the control HBV infection via the production of IFN-γ and non- cytolytic viral clearance (Kakimi, Guidotti et al. 2000), their contribution in the early stages of natural infection remains controversial. NKT cells recognise TCR restricted lipid antigens in the context of CD1d and produce cytokines rapidly following antigen recognition that bear broad effects on the activation of NK cells as well as T and B cells (Tupin, Kinjo et al. 2007).
An early activation of NKT cells has been reported in both animal models and HBV patients
28 at the very early time points after infection (Guy, Mulrooney-Cousins et al. 2008) (Fisicaro,
Valdatta et al. 2009). Animal studies have further highlighted a role for NKT cells in HBV- induced hepatitis (Vilarinho, Ogasawara et al. 2007) (Baron, Gardiner et al. 2002). However a new study has demonstrated that NKT cells also contribute to viral control and the emergence of adaptive immunity. In an adenovirus HBV mouse model, which delivers a replication competent HBV genome surmounting the non-permissiveness of murine hepatocytes to HBV, hepatocytes use secretory phospholipases i.e microsomal triglyceride transfer protein (MTP) and CD1d to detect the presence of HBV (Zeissig, Murata et al. 2012).
Through the display of modified self-lipids on the surface of infected hepatocytes they alert
NKT cells to generate/prime an HBV-specific immune response (Zeissig, Murata et al. 2012).
Future studies in HBV patients are warranted to affirm a central role of NKT cells in human infection with HBV. Whether NK cells play an active role in the containment of acute HBV infection and their potential role in persistent infection is one of the main objectives of this work and will be considered further in chapter 3.
Adaptive immune response
Crucial to the immunity against cytopathic viruses are CD8+ T cells. Previous elegant studies have substantiated their central role in HBV clearance in both chimpanzee and human infection (Thimme, Wieland et al. 2003) (Maini, Boni et al. 2000). T cell mediated control via non-cytolytic pathways and production of cytokines such as IFN-γ and TNF-α, which potently abolish HBV replication, have been supported by findings in the transgenic mouse model but also in a cell culture model (Guidotti, Ishikawa et al. 1996) (Phillips, Chokshi et al. 2010).
However, virus-specific CD8+ T cells are also capable of mediating viral clearance by lysis of a proportion of virus-infected hepatocytes, which may in turn contribute to liver inflammation.
A clear correlation exists between a vigorous and multi-specific CD8+ T cell response and
29 HBV clearance, whereas the characteristic feature of persistent infection is a dysfucntional, weak and monospecific T cell response (Bertoletti and Ferrari 2012).
What are the factors that contribute to the T cell collapse in CHB infection? A number of defects have been described to account for the observed quantitative and qualitative deficiencies observed in HBV-specific CD8+ T cells. In addition to weak and transient T cell responses with narrower specificity, chronically infected patients have characteristically low numbers of virus-specific T cells (Rehermann, Fowler et al. 1995). Gene expression profiling identified an upregulation of a pro-apoptotic member of the Bcl-2 superfamily, Bim, which enhances HBV-specific CD8+ T cell vulnerability to death (Lopes, Kellam et al. 2008). Multi- specific functional responses could be restored following downstream blockade of Bim, identifying it as a potential target for immunotherapy. The apoptotic propensity and premature elimination of these cells is likely compounded by defective antigen presentation in the liver (Maini and Schurich 2010) (Bowen, Zen et al. 2004).
In patients unable to control viral replication, the remaining HBV-specific T cells are functionally inactivated or exhausted displaying reduced proliferative potential and impaired
IFN-γ production (Das, Hoare et al. 2008) (Schurich, Pallett et al. 2013). The influence of viral load and constant antigenic stimulation on gradual functional regression of virus-specific T cells has been previously characterised in animal models of persistent viral infections such as lymphocytic choriomeningitis virus (LCMV) (Wherry, Blattman et al. 2003) (Wherry 2011).
Likewise in CHB, intrahepatic HBV-specific CD8+ T cells are inversely proportional to HBV
DNA levels (Fisicaro, Valdatta et al. 2010). Virus-specific T cells for the major HBV epitopes are barely detectable at viral loads exceeding 107 copies/mL, presumably having succumbed to repeated cycles of T cell activation (Webster, Reignat et al. 2004). Equally long-term exposure to HBV viral antigens could further impact on T cell function. This is supported by
30 the fact that antiviral drugs, despite successfully suppressing HBV replication, only have a modest effect on the production of the sub-viral particles achieving transient HBV-specific T cell responses (Boni, Penna et al. 2003). Moreover, disproportionate co-inhibition has been demonstrated to drive exhaustion and potentially encourage Bim-mediated apoptosis of virus specific CD8+ T cells (Maini and Schurich 2010). A number of candidate inhibitory receptors such as programmed death-1 (PD-1) and cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) are upregulated on HBV-specific CD8+ T cells in CHB (Boni, Fisicaro et al.
2007) (Schurich, Khanna et al. 2011). The therapeutic potential of PD-1 blocking has been shown in trials in simian immunodeficiency virus (SIV) and HCV but also in the HBV mouse model (Velu, Titanji et al. 2009) (Maier, Isogawa et al. 2007). Although this is a promising therapeutic approach, co-expression of additional inhibitory molecules on CD8+ T cell populations such as T cell immunoglobulin mucin-3 (Tim-3) or 2B4, necessitate a more detailed understanding of the degree of synergy and redundancy of the various pathways
(Nebbia, Peppa et al. 2012) (Raziorrouh, Schraut et al. 2010) (Fig. 1D).
Figure 1D. Schematic representation of molecular defects. Complex layers of co-inhibition drive T cell exhaustion in chronic HBV infection (adapted from Maini and Schurich 2010)
31 These intrinsic defects can be perpetuated by extrinsic factors present in the liver microenvironment. In contrast to sterile organs such as the spleen, the liver is an immunologically distinct organ that is constantly exposed to innocuous gastrointestinal antigens. To adapt to its physiological function, a locally regulated immune system has evolved to efficiently eliminate pathogens, while tolerating the large number of gastrointestinal tract derived antigens (Protzer, Maini et al. 2012). T cell exhaustion and attrition by apoptosis may be encouraged in the liver following tolerogenic activation by hepatocytes and liver sinusoidal endothelial cells (LSECs) (Protzer, Maini et al. 2012).
Additionally, the liver nutrient milieu and in particular depletion of the levels of the amino acid arginine, can result in reduced or defective intracellular signalling cascades following engagement of T cell receptor (Das, Hoare et al. 2008). Decreased interleukin-2 (IL-2) production and high levels of the immunosuppressive cytokines IL-10 and transforming growth factor-β (TGF-β) can further impair the maintenance of robust T cell responses
(Tinoco, Alcalde et al. 2009).
CD4+ T cells have been shown to be particularly important in determining the magnitude and quality of the subsequent CD8+ T cell response to HBV (Asabe, Wieland et al. 2009).
However, priming of CD4+ T cells in the liver has been noted to lead to poor production of
TH1 cytokines (Maini and Schurich 2010) (Knolle, Schmitt et al. 1999). Mirroring HBV-specific
CD8+ T cells, chronically infected patients also have a narrower repertoire of virus-specific
CD4+ T cells with reduced proliferative capacity and ability to secrete cytokines (Ferrari,
Penna et al. 1990; Bertoletti and Ferrari 2012). Regarding regulatory populations (Tregs), their precise role remains controversial in CHB; some studies have suggested that they can also hamper the ability of T cells to expand and survive (Manigold and Racanelli 2007).
Although available data on the frequencies of classical regulatory CD4+CD25highFoxp3+ cells in CHB have been conflicting (Franzese, Kennedy et al. 2005) (Xu, Fu et al. 2006) (Peng, Li et
32 al. 2008) their depletion increased the function of HBV-specific T cells. However, a similar effect was observed in patients with resolved HBV infection (Franzese, Kennedy et al. 2005).
In addition to T cells being a central component required for HBV clearance, B cells play an important role in inducing a protective humoral immune response. HBsAg-specific antibodies are neutralising (Rehermann and Nascimbeni 2005) and production of anti- envelope antibodies correlates temporally with clearance of HBV (Alberti, Diana et al. 1978).
The presence of HBsAg-specific and HBcAg-IgG antibodies are associated with lifelong protection against inection/reinfection in the host. However, in chronic infection the failure of the cellular arm affects the activation and protective efficacy of the humoral arm. Besides antibody production, B cells in mice have been found to have pleiotropic functions including the ability to regulate immune responses through production of cytokines such as IL-10
(Fillatreau, Sweenie et al. 2002) (Mauri, Gray et al. 2003) (Mizoguchi, Mizoguchi et al. 2002).
More recently the presence of a phenotypically distinct IL-10 producing subset of B cells
(Breg) has been described in chronic infection with HBV, suppressing HBV-specific CD8+ T cell responses (Das, Ellis et al. 2012).
Of interest, Mary Carrington’s group reported an association between higher levels of HLA-
DP surface expression, but also mRNA levels, and the 496GG genotype correlating with HBV persistence (Thomas, Thio et al. 2012). This novel variant showed a more robust link with
HBV disease outcome compared to the single nucleotide polymorphisms (SNPs) reported by the previous Asian studies (Kamatani, Wattanapokayakit et al. 2009). HLA-DP is expressed on the surface of APCs, including B cells, and plays an important role in presentation of antigens to CD4+ T cells, instructing both humoral and cellular immunity. A working hypothesis was put forward suggesting that the elevated expression of HLA-DP may be promoting a TH2 response leading to HBV persistence. However, the question how the
33 expression of this molecule in the HBV infected liver is influencing immunity, in terms of
CD4+ T cell and B cell responses to HBV, remains unclear at present.
Recently new evidence has emerged suggesting the direct involvement of NK cells in the negative regulation of effector T cell responses in the mouse LCMV infection (Waggoner,
Cornberg et al. 2012) (Lang, Lang et al. 2012). In the model proposed by Welsh and
Waggoner, viral pathogenesis varies according to the infecting dose of LCMV clone 13 and the presence of NK cells. High dose clone 13 LCMV infection leads to persistent infection accompanied by T cell exhaustion, whilst at lower doses the virus is cleared. At high dose infection the presence of NK cells is of benefit to the host curtailing excessive T cell responses, whereas at low viral loads NK cell contribution is not noteworthy given that the antiviral T cell response is sufficient to establish control in the absence of immunopathology.
However, infection with an intermediate dose of clone 13, leads to severe pathology in the lungs and liver of infected animals mediated by T cells, suggesting that NK cell control of T cells is disadvantageous in this setting impairing viral control. Along these lines, depletion of
NK cells resulted in stronger T cell responses, which effectively altered the viral set point dictating whether pathology, viral persistence or clearance will develop (Welsh and
Waggoner 2013). The ability of NK cells to modulate the antiviral response in CHB is not known. Their immunoregulatory role will be explored and discussed further in chapter 4, focusing on the potential of NK cell depletion to resurrect T cell responses encouraging viral clearance.
Immunopathology of CHB and NK cells – Multiple hats for NK cells?
Liver inflammation and immunopathology is the result of the repeated efforts of the immune response to curb viral replication. In contrast to patients with HCV infection, CHB patients experience necroinflammatory flares. Initially, it was thought that the action of
34 virus-specific CD8+ cytotoxic T lymphocytes (CTL) was solely responsible for the cytolytic destruction of HBV infected hepatocytes. However, early work from the HBV transgenic mouse model alluded to a potential involvement of the non-specific infiltrate recruited in the liver at the height of inflammation (Ando, Moriyama et al. 1993). Subsequent studies in human CHB demonstrated that equivalent numbers of intrahepatic virus-specific CD8+ T cells were associated with either protection or pathology (Maini, Boni et al. 2000).
Moreover, an influx of non-antigen specific lymphocytes was observed in patients with biochemical evidence of liver inflammation (raised ALT), highlighting their potential role in liver disease (Maini, Boni et al. 2000). In the transgenic mouse model, CTL production of IFN-
γ induced recruitment of mononuclear cells, which in turn recruited NK1.1+CD3- NK cells with a 10-12 fold increase in their numbers in the inflammatory infiltrate compared to baseline (Sitia, Isogawa et al. 2004) (Kakimi, Lane et al. 2001). Importantly, chemokine blockade diminished the influx of non-antigen specific lymphocytes and ameliorated the severity of liver disease, without affecting non-cytolytic clearance of the virus (Sitia, Isogawa et al. 2004) (Sitia, Isogawa et al. 2002) (Kakimi, Lane et al. 2001). An infiltration of NK cells has also been shown to contribute to hepatotoxicity in a number of different models, including Pseudomonas induced hepatotoxicity (Muhlen, Schumann et al. 2004), murine cytomegalovirus (MCMV) induced hepatitis in mice (Salazar-Mather, Orange et al. 1998), and in liver disease with persistent HCV infection (Nuti, Rosa et al. 1998). NK cells may be, therefore, a candidate cell type for mediating liver pathology in humans, as they are abundant in the cellular infiltrate and account for a large proportion of resident intrahepatic lymphocytes (Doherty, Norris et al. 1999) (Norris, Collins et al. 1998).
Given that hepatocytes may be relatively resistant to perforin/granzyme-mediated cytotoxicity (Tay and Welsh 1997; Kafrouni, Brown et al. 2001), a role for death receptor pathways and members from the TNF superfamily inducing hepatocyte apoptosis has been
35 previously suggested (Galle, Hofmann et al. 1995; Balkow, Kersten et al. 2001; Zheng, Wang et al. 2004) (Fig. 1E, potential pathways of liver injury). Dysregulated apoptosis is increasingly recognised to play a part in the initiation of hepatic inflammation (Malhi and
Gores 2008). It is has been further postulated that persistent hepatocyte apoptosis contributes to fibrogenesis, chronic liver failure, and even development of hepatocellular carcinoma (HCC) (Malhi and Gores 2008). Human studies from our lab have implicated the
TNF-related apoptosis-inducing ligand (TRAIL) in liver inflammation (Dunn, Brunetto et al.
2007). Whereas healthy hepatocytes are sensitive to FasL, they are normally protected against TRAIL-induced apoptosis (Gores and Kaufmann 2001). However, HBV infection may alter hepatocyte sensitivity to TRAIL mediated death (Janssen et al., 2003; Liang et al., 2007).
In support of this, increased expression of TRAIL-R2 on the surface of hepatocytes in liver biopsies from patients with CHB has been previously reported (Dunn, Brunetto et al. 2007).
In the same study, NK cells were found to upregulate TRAIL and contribute to hepatocyte injury during disease flares in eAg- CHB patients (Dunn, Brunetto et al. 2007). Different cytokines have been shown to upregulate TRAIL expression on NK cells including type I IFNs,
IL-2 and IFN-γ (Ishiyama, Ohdan et al. 2006) (Tu, Hamalainen-Laanaya et al. 2011; Sarhan,
D'Arcy et al. 2012). Equally, hepatocytes can become vulnerable to TRAIL mediated death following modulation of death receptor expression encouraged by cytokines such as IFN-
α and IL-8 during active HBV infection (Dunn, Brunetto et al. 2007) or deletion of NF-kappa-B essential modulator (NEMO), the regulatory subunit of the IKK complex mediating NF-kappa-
B activation (Beraza, Malato et al. 2009). The observation that NK cell mediated killing of hepatocytes was not completely blocked by TRAIL blocking antibodies suggested that NK cells may employ additional ligands to mediate liver damage, such as the Fas receptor
(CD95)/Fas ligand(CD95L) interaction. The importance of the Fas pathway and its role in hepatocyte apoptosis, as well as fulminant hepatitis, has been previously proposed by several studies (Ryo, Kamogawa et al. 2000) (Bortolami, Kotsafti et al. 2008) (Ogasawara,
36 Watanabe-Fukunaga et al. 1993) (Mochizuki, Hayashi et al. 1996) (Hayashi and Mita 1999).
More recently Okazaki et al provided evidence that dendritic cell (DC)-activated NK cells are capable of inducing HBV-infected hepatocyte degeneration in the urokinase-type plasminogen activator/severe combined immunodeficiency (uPa/SCID) mouse through the
Fas/FasL system (Okazaki, Hiraga et al. 2012). Supporting a role for Fas mediated liver injury, highly activated liver NK cells were found to be cytotoxic to murine hepatitis virus-3 (MHV-3) infected hepatocytes, an effect that was inhibited by anti-Fas ligand (FasL) plus anti-NKG2D mAbs (Zou, Chen et al. 2010). In the same study there was accumulation of hepatic NK cells in HBV patients with acute liver failure. Disease progression also correlated with increased levels of expression of FasL and the natural cytotoxicity receptors (NKp30 and NKp46) on peripheral NK cells (Zou, Chen et al. 2010). A larger study assessing the role of hepatic NK cells in CHB demonstrated that NK cells expressing activation receptors (NKp30, NKp44) preferentially accumulated in the livers of patients with active disease and correlated with liver injury (Zhang, Zhang et al. 2011). However, a direct role for NK cells in mediating liver damage via the Fas pathway or indeed the natural cytotoxicity receptors has not been fully established in human infection with HBV. NK cells are also a potent source of the pro- inflammatory cytokine TNF-α that has been shown to augment liver injury (Mizuhara, O'Neill et al. 1994). Interestingly there is co-operation between the extrinsic (death receptor) pathway of cell death and the intrinsic (or mitochondrial pathway) as in the case of TNF-
α and FasL activating Bcl-2 interacting-domain death agonist (Bid) and Bim (Schmich,
Schlatter et al. 2011). Hepatic TNF and TNF-receptor 1 (TNFR1) expression are enhanced in
CHB (Fang, Shen et al. 1996) and susceptibility of hepatocytes to TNF-induced death has been shown to be modulated by viral infection(Wohlleber, Kashkar et al. 2012), but again their potential involvement in liver damage and contribution of NK cells remains unclear .
37 Another interesting feature of liver injury is hepatocyte expression of stress ligands for the
NK receptor NKG2D, which can override weaker inhibitory signals. In the context of CHB
‘stressed’ hepatocytes may provoke their own destruction. Data from the transgenic mouse model demonstrated that HBV infection can induce NKG2D ligands (Rae1 and MULT1), sensitising hepatocytes to NKG2D-mediated killing (Chen, Wei et al. 2007), whereas knocking down multiple NKG2D ligands on hepatocytes was able to protect against fulminant hepatitis (Huang, Sun et al. 2012). The role of NKG2D and its ligands in human chronic HBV infection is an area that requires further investigation (Fig. 1E).
Hepatocyte)
Caspase)Cascade) Lysis))) of)target)cell)
TRAIL4R2) Fas) NKG2DL) TNFR1) ! TNF4α" TRAIL! FasL) NKG2D) NK)cell)
Figure 1E. Potential receptor/ligand interactions in the liver and potential mechanisms of injury
These observations need to be reconciled with the potential anti-fibrotic effect of NK cells and the potential critical role of NK cells in tumour protection in the setting of CHB
(Kamimura, Yamagiwa et al. 2012) (Radaeva, Sun et al. 2006). Studies from rodents have highlighted that NK cells may inhibit liver fibrosis. This effect is mediated via TRAIL and
NKG2D dependent killing of hepatic stellate cells (HSC), key mediators of fibrosis, and production of IFN-γ that inhibits HSC activation (Taimr, Higuchi et al. 2003) (Radaeva, Sun et al. 2006). More recently NKp46 mediated recognition and killing of human and mouse
38 hepatic stellate cells was reported to ameliorate liver fibrosis (Gur, Doron et al. 2012). In support of this role, it has been suggested that IFN-α therapy may mediate anti-fibrotic effects through its potential to augment NK cell mediated killing of stellate cells in HCV infection (Glassner, Eisenhardt et al. 2012). Equally TRAIL mediated killing of infected hepatocytes may constitute an important mechanism of antiviral defence transpiring at the expense of liver damage. It is conceivable that continuous rounds of viral replication and NK cell activation in the liver, contributing to hepatocellular damage and subsequent fibrosis, dominate over their potential anti-fibrotic effect. The lack of NK cells in fibrotic areas favours progression, whilst TGF-β production by activated HSCs (Canbay, Friedman et al.
2004) may be effectively suppressing NK cell function to accelerate liver fibrosis. Thus a complex picture is emerging where the balance of cytokines dictates the predominant function of NK cells. Whether specific subsets of NK cells in CHB serve anti-fibrotic and/or hepatoprotective versus pathogenic functions remains unclear.
NK cells are therefore emerging as innate effectors with a dual role during HBV infection.
How exactly the cytokine milieu and viral components combine to enhance NK cell activation and sensitise hepatocytes to cell death requires further exploration. Further studies are required to better understand the factors triggering and mediating the opposing roles of NK cells in CHB to allow these to be successfully exploited for therapeutic targeting.
NK cells general aspects
NK cells are enriched in the human liver, an organ with a predominant innate immunity, comprising 40-60% of the intrahepatic lymphocyte pool, more than 3 fold higher than in the periphery (Doherty and O'Farrelly 2000) (Doherty, Norris et al. 1999) (Gregoire, Chasson et al. 2007). The predominance and constitutive activation of NK cells in the liver sinusoid highlights their potential for an important role in the defence against hepatotropic viruses
39 such as HBV.
NK cells were initially described as large granular lymphocytes belonging to the innate arm of the immune response endowed with the functional ability to kill target cells ‘naturally’, without any priming and not restricted by the target’s cell expression of MHC (Kiessling,
Klein et al. 1975) (Lanier, Phillips et al. 1986). Since entering the immunological scene in the mid 1970’s, it has become apparent that NK cells are more than just natural ‘killers’ with constitutive cytolytic functions. The composite modern day NK cell likely resides at the crossroads of innate and adaptive immunity, displaying a multitude of functions that help co-ordinate immune responses against invading pathogens, in addition to tumour immunosurveilance (Vivier, Raulet et al. 2011) (Vivier, Tomasello et al. 2008). This is achieved via virtue of expression of a wide array of NK cells receptors that are finely attuned to ensure self-tolerance, while permitting effective responses against microbial assaults and tumour transformation. Recent advances in NK cell biology highlighted that NK cells share many features normally associated with adaptive immunity such as the capacity for memory and tolerisation (Coudert, Zimmer et al. 2005; Oppenheim, Roberts et al. 2005); (Raulet and
Vance 2006; Tripathy, Keyel et al. 2008) (Sun, Beilke et al. 2009) (Paust and von Andrian
2011). Moreover, it is now appreciated that NK cells adapt to the environmental milieu, which can potentially modify NK cell function, resulting in ignorance or exhaustion (Di Santo
2008). In the following sections general aspects of NK cell biology including development and differentiation, regulation of NK cell activation, as well as a general overview of the antiviral potential of NK cells will be discussed.
NK cell developmental origins and subsets
NK cells represent the third major lineage of lymphocytes alongside the T and B cell lineages
40 (Di Santo 2006). Traditionally NK cells are defined phenotypically by the absence of CD3 and expression of CD56, the 140kDa isoform of neural cell adhesion molecule (NCAM) (Lanier,
Testi et al. 1989). However, murine NK cells do not express CD56. Instead, NKp46 has been suggested as the most specific phenotypic NK cell marker across mammalian species
(Walzer, Jaeger et al. 2007), although it can be found on a small subset of T lymphocytes as well as some gut innate lymphoid cells (ILCs) subsets (Walzer, Jaeger et al. 2007) expressing the retinoic acid receptor-related orphan receptor γt (RORγt) transcription factor (Sanos, Bui et al. 2009) (Satoh-Takayama, Vosshenrich et al. 2008) (Luci, Reynders et al. 2009). These latter ILCs, both in human and mouse, are functionally distinct from conventional NK cells, produce interleukin-22 (IL-22), are non-cytotoxic and are abundantly represented in mucosal tissues, particularly of the gut (Spits and Di Santo 2011). Some human NK cells lack or have low-level expression of NKp46, but the nature and precise function of this subset is not fully determined.
NK cell development from hematopoietic stem cells is a carefully orchestrated process involving discrete stages; CD34+ hematopoietic stem cells differentiate into common lymphoid progenitors that give rise to NK cell precursors that then develop into immature
NK cells. The final maturation step into fully functional NK cells involves the acquisition of activating and inhibitory receptors that control NK cell effector functions (Lanier 2005). Both lineage specifying and extrinsic signals are required to guide the cells along distinct differentiation pathways (Di Santo 2009). The basic leucine zipper transcription factor E4BP4
(also known as NFIL3) has been identified as an NK lineage specification factor (Gascoyne,
Long et al. 2009), whereas inhibitor of DNA binding 2 (ID2) and GATA3 have been identified as downstream targets of E4BP4 regulating the production of mature NK cells from immature NK (iNK) cells (Male, Nisoli et al. 2012) (Luevano, Madrigal et al. 2012). However, it has become apparent that the transcriptional network of NK cells is organised in a far
41 more complicated fashion than was initially envisaged. Several other transcription factors have been identified but it remains unresolved at what exact stage they act to influence the lineage fate of NK cells (Huntington, Nutt et al. 2013).
It is generally accepted that NK cells develop primarily in the bone marrow, suggested by early studies of selective bone marrow ablation in mice (Haller and Wigzell 1977) (Haller,
Kiessling et al. 1977). Bone marrow-derived human CD34+ haematopoietic precursor cells
(HPCs) can develop into cytolytic NK cells in vitro and at least the very early stages of NK cell development are dependent on bone marrow stromal elements such as IL-7, IL-2, IL-15, stem cell factor and FLT3 ligand (Di Santo 2006). The cytokine IL-15 has been found to be critical for the development and maturation of NK cells and mice deficient in IL-15 or IL-15 receptor alpha-chain (IL-15Ra) display a striking reduction of NK cell numbers (Kennedy,
Glaccum et al. 2000) (Lodolce, Boone et al. 1998; Sun and Lanier 2011) (Di Santo 2006).
However, complete in situ characterization of the NK cell developmental pathway from
CD34+ HPCs within the bone marrow is still lacking in humans, raising the possibility that NK cell precursors can traffic to distinct peripheral anatomical locations for their final stages of maturation. Recent examination of NK cell ontogeny suggested that immune precursor cells with the potential to develop into NK cells can be found in human secondary lymphoid tissues (SLT) and in other distinct anatomical locations including the liver (Eissens, Spanholtz et al. 2012) (Male, Hughes et al. 2010) (Freud and Caligiuri 2006) (Freud, Yokohama et al.
2006) (Freud, Becknell et al. 2005) (Moroso, Famili et al. 2011). Whether these NK cell populations represent separate lineages or denote predominately less mature peripheral cells that have originated from the bone marrow remains incompletely defined. Unlike the well established stages of T cell in the thymus, NK cell development and differentiation has been ascribed several arbitrary stages on the basis of sequential acquisition of NK cell specific markers and functional capabilities (Freud, Becknell et al. 2005) (Freud and Caligiuri
42 2006) (Kim, Iizuka et al. 2002) (Sun and Lanier 2011). The most complete pathway of NK cell differentiation reported in humans takes place in secondary lymphoid tissue (SLT) encompassing four developmental stages, specified by the expression of CD34, CD117 (c- kit), and CD94. These include: stage 1 pro-NK (CD34+CD117−CD94−); stage 2, pre-NK,
(CD34+CD117+CD94−); stage 3, immature iNK, (CD34−CD117+CD94−); and stage 4
(CD34−CD117+/−CD94+) NK cells, corresponding to CD56bright subset (Freud, Yokohama et al.
2006) (Fig. 1F).
The total population of mature NK cells, representing 10% of peripheral blood lymphocytes, is phenotypically and functionally very diverse (Caligiuri 2008). Different NK cell subsets can be found in lymphoid and non-lymphoid organs including the liver, lung, thymus, pancreas and uterus (Freud and Caligiuri 2006) (Colucci, Caligiuri et al. 2003). Traditionally in humans, mature NK cells are divided into two main subsets depending on the density of CD56 expressed on their cell surface (Cooper, Fehniger et al. 2001). In peripheral blood, bone marrow, and spleen, CD56dim NK cells predominate, representing around 90% of total NK cells, whereas tonsils and SLT are enriched for CD56bright NK cells. The CD56bright subset lacks expression or expresses low levels of the low affinity Fc-receptor CD16, has low expression of inhibitory killer cell immunoglobulin-like receptors (KIRs) and of cytotoxic molecules such as perforin and granzymes (Cooper, Fehniger et al. 2001). NK cell CD56bright are uniformly positive for inhibitory CD94/NKG2A, express the high affinity IL-2 receptor α chain (CD25), as well as CD62L and CCR7 that aids selective trafficking to the lymph nodes. On the other hand the CD56dim NK cell subset is CD16+, displays a variegated expression of KIRs, NKG2A, CD62L, and contains intracellular granules with large amounts of perforin and granzymes (Cooper,
Fehniger et al. 2001). It is now generally accepted that the CD56dim subset represents the more mature phenotypically subset (stage 5) deriving directly from the CD56bright subset, consistent with Lanier’s original proposition and more recent conclusive studies (Caligiuri
2008) (Romagnani, Juelke et al. 2007) (Shilling, McQueen et al. 2003) (Huntington, Legrand
43 et al. 2009) (Fig. 1F). Progression from CD56bright to CD56dim is likely part of a continuum in their development, with intermediates of this process characterised by a gradual loss of
NKG2A paralleled with a sequential acquisition of KIRs and CD57, as a molecule expressed on terminally differentiated NK cells (Caligiuri 2008) (Juelke, Killig et al. 2010) (Bjorkstrom,
Riese et al. 2010). A stage-wise differentiation program has also been suggested for murine
NK cells based on surface expression of CD11b and CD27 (CD11blowCD27low →
CD11blowCD27high → CD11bhighCD27high → CD11bhighCD27low). CD11blowCD27high murine NK cells share some characteristics with CD56bright NK cells, whereas the CD11bhighCD27low subset is more similar to CD56dim NK cells (Chiossone, Chaix et al. 2009) (Hayakawa and Smyth
2006) (Hayakawa, Andrews et al. 2010). Of note the expression of TRAIL defines murine foetal NK cells (NK1.1+TRAIL+CD49b-Mac1-Ly49-). Although this population persists in the liver into adulthood its size is reduced as conventional NK cells (NK1.1+CD49b-Mac1+Ly49+) develop, representing the majority of the intrahepatic population in adult mice (Takeda
2005 Blood). The lack of Ly49 expression on neonatal NK cells has been subsequently linked to restricted induction of Eomes within the liver environment, whereas the immature pool of CD49b-TRAIL+ NK cells appears to be dependent on T-bet (Gordon S Immunity 2011). How exactly these molecular events are regulated and the significance of distinct hepatic NK cell repertoires with potentially unique functions remain unresolved.
44 Bone'Marrow' Secondary'Lymphoid'Hssue' Blood' HPC' Pro$NK' Pre$NK' iNK' NK' mNK' CD56brightCD16$' CD56dimCD16$'
Stage'1' Stage'2' Stage'3' Stage'4' Stage'5' CD34$' CD117+/$' CD117$' CD117+' NKp46+' NKp46+' CD161+' CD94/NKG2A+' CD94/''''''' CD94/NKG2A$' CD16$' NKG2A+/$' ' KIR$' CD16+' KIR+
Figure 1F. Model of human NK cell development (adapted from Caligiuri 2008)
As demonstrated by their existence in both humans and mice, the presence of functionally heterogenous NK cell subsets has been conserved through species. What is the evolutionary advantage and biological importance of having NK cells with distinct effector functions, tissue distribution and varied states of differentiation? Although the answers to these questions continue to be the focus of investigation, this diversity could provide a means to adapt to different conditions and respond rapidly to multiple types of assault. Overcoming methodological shortcomings and developing new assays allowing in situ analysis will increase our knowledge of NK cell development, normal tissue localisation and homeostatic trafficking.
NK cell receptors and education
NK cells express an armory of germline encoded activating and inhibitory receptors that do
45 not undergo somatic recombination (Lanier 2005) (Bryceson, March et al. 2006). Three major super-families of natural killer receptors (NKR) have been described: the killer cell immunoglobulin-like receptors (KIR) superfamily which recognises classical major histocompatibility complex (MHC) class I molecules (HLA A, B and C), the C-type lectin superfamily recognising non-classical MHC class I or class I-like molecules, and the natural cytotoxicity receptors (NCR). Although, there is some evidence that NCRs recognise viral haemagglutinins (Mandelboim, Lieberman et al. 2001) their ligands remain poorly defined with the exception of B7-H6 that was identified as a ligand for NKp30 (Brandt, Baratin et al.
2009). Other NKR that function primarily as co-receptors have been described but their ligands and significance remain unknown in some cases (Bryceson, Chiang et al. 2011) (Table
1.1).
Ac5va5ng*Receptors,** Inhibitory*Receptors* adhesion*or*co8s5mula5on* * Receptor* Ligand* Receptor* Ligand* CRACC& CRACC& KIR$L& HLA$C,&B&and&A& A1b1& VCAM$1& LAIR$1& Collagen& Integrin& CD94$NKG2A& HLA$E& B2&Integrin& ICAM$1& KLRG1& Cadherins& DNAM& CD112,&CD155& NKP$R& LLT$1& CD16& IgG& *CD244& CD48& NKp46& Viral&haemaggluLnins& NKp44& Viral&haemaggluLnins& NKp30& B7H6& NKP80& AICL& NKG2D& MICA/B,&ULBP1$6& CD96& CD155& CD94$ HLA$E& NKG2C/E& KIR$S& ?HLA$C&
Table 1.1 Illustration of the NK cell interaction repertoire in humans. Cytokines, chemokines and their receptors are not depicted in this table. *CD244(2B4) can act as an activating or inhibitory molecule. KIR, killer cell immunoglobulin-like receptors; LAIR; leucocyte associated immunoglobulin
46 like receptor; KLRG-1, killer cell lectin-like receptor G1; NKR-P1, NK cell receptor protein 1; LLT, lectin like transcript; HLA, human leucocyte antigen; CRACC, CD2-like receptor activating cytotoxic cell; VCAM-1, vascular cell adhesion molecule 1; ICAM, intracellular adhesion molecule.
There are many more activating receptors expressed by each NK cell compared to the inhibitory receptors. The proximal signaling from these activating receptors occurs through highly divergent signaling domains/motifs and their association with different adaptor proteins. For instance, the activating receptors CD16, the C-lectin NKG2C/CD94, or natural cytotoxicity receptors (NKp30, NKp44, and NKp46) interact with immunoreceptor tyrosine- based activating motif (ITAM)- containing adaptors such as CD3ζ, DAP12, FcRγ that recruit spleen tyrosine kinase (SYK) and zeta chain associated protein kinase of 70 kda (ZAP70) to mediate signaling (Lanier 2008) (Vivier, Nunes et al. 2004). Other receptors, such as NKG2D a
C-type lectin-like homodimer, that recognises stress induced ligands or the SLAM family of receptors such as 2B4, use an alternative signaling mechanism involving non-ITAM-bearing adaptor proteins e.g. DNAX-activating protein of 10 kD (DAP10) and SAP respectively
(Bryceson, March et al. 2006) (Chiesa, Mingueneau et al. 2006) (Lanier 2005). The advantage of using several different activating pathways is unclear but may reflect the requirement for the summation or synergy of activating signals to generate efficient NK cell responses
(Bryceson, March et al. 2006) (Lanier 2003). Despite the use of various signaling molecules, activation of phospholipase C-γ (PLC-γ) and Vav proteins by multiple activating receptors suggests their non-redundant role in Ca2+ mobilisation and NK cell mediated target cytotoxicity (Lanier 2008).
NK cell inhibitory receptors are mostly members of the immunoglobulin (Ig) superfamily of receptors or lectin-like i.e the CD94-NKG2A heterodimer, and recognise classical or non- classical MHC class I molecules, and are heterogeneously expressed on resting NK cells. In
47 particular KIRs display significant allelic and haplotypic diversity (Shilling, Guethlein et al.
2002) (Yawata, Yawata et al. 2008). Compared to the activation receptors, inhibitory receptors have conserved features with respect to their signaling and specificity. A common feature of the inhibitory receptors is the possession of at least one intracellular, immunotyrosine-based inhibitory motif (ITIM) present in their cytoplasmic tail (Lanier 2005)
(Chiesa, Mingueneau et al. 2006) (Bryceson, March et al. 2006) (Long 2008). Recruitment of the Src homology 2 domain-containing phosphatase 1 (SHP-1) is required for inhibition by an
ITIM–containing receptor inducing dephosphorylation of signaling molecules i.e Vav1 (Long
2008).
NK cells have developed an elaborate detection system where the reciprocal antagonism of activating and suppressing signals from their cell surface receptors maintains a dynamic equilibrium regulating NK cell activation. Inhibitory receptors are employed by NK cells to assess the absence of constitutively expressed self-molecules on susceptible target cells. The
‘missing self’ hypothesis was originally proposed by Kärre and colleagues in a seminal study, where NK cells were shown to be able to kill MHC class I negative tumor cells, yet sparing their MHC class I expressing counterparts, predicting the existence of inhibitory receptors on
NK cells (Karre, Ljunggren et al. 1986). However, NK cells are not only able to evaluate the
‘absence’ of MHC class I ensuring tolerance to self, they are also reliant on inhibitory signaling through MHC class I during their development and differentiation in order to become fully competent (Hoglund and Brodin 2010). Analogous to T lymphocytes, NK cells are educated to self versus altered-self discrimination/recognition, however, the molecular strategies regulating this process differ (Sun and Lanier 2011). Evidence for such MHC- dependent functional maturation of NK cells was initially derived from studies of MHC class I deficient mice, where NK cells were found to be hyporesponsive (Liao, Bix et al. 1991)
(Ljunggren, Van Kaer et al. 1994) (Hoglund, Ohlen et al. 1991). Additional work from both
48 human and animal studies demonstrated that NK cells lacking expression of inhibitory receptors, as well as NK cells expressing inhibitory receptors with no self-ligand present in the host, exhibit an overall inert phenotype (Fernandez, Treiner et al. 2005) (Kim, Poursine-
Laurent et al. 2005) (Anfossi, Andre et al. 2006). Thus, for an NK cell to become functionally competent, it needs to express at least one inhibitory receptor that recognises a self-ligand present in the host i.e the ligands for inhibitory MHC class I–specific receptors, such as
KIR2DL1/2, KIR3DL1, and NKG2A in humans, and Ly49 receptors in mice (Kim, Sunwoo et al.
2008) (Kim, Poursine-Laurent et al. 2005) (Anfossi, Andre et al. 2006) (Andersson, Fauriat et al. 2009) (Fauriat, Andersson et al. 2008). This education, also referred to as ‘tuning, licensing or arming’ ensures the maturation of a NK cell functional repertoire in a quantitative manner, which is adjusted to the self-MHC class I environment following a tunable rheostat model (Fernandez, Treiner et al. 2005) (Kim, Poursine-Laurent et al. 2005)
(Anfossi, Andre et al. 2006) (Raulet and Vance 2006) (Brodin, Karre et al. 2009) (Brodin,
Lakshmikanth et al. 2009) (Joncker, Fernandez et al. 2009) (Hoglund and Brodin 2010). The alternative ‘disarming’ hypothesis suggests that all NK cells are by default responsive, but if unimpeded by MHC class I-specific inhibitory receptors, chronic stimulation through activating receptors renders NK cells anergic (Gasser and Raulet 2006). Similar to exhausted
T cells recovering functionality following cytokine stimulation or blockade of the tolerising signal (Barber, Wherry et al. 2006) (Teague, Sather et al. 2006), tolerance of unlicensed or disarmed NK cells can be broken in the presence of inflammatory cytokines or infection or local inflammatory conditions (Yokoyama and Kim 2006; Orr and Lanier 2010) (Sun and
Lanier 2008).
With advances in nanoscale imaging it has been possible to demonstrate that education by
MHC class I recognition leads to dramatic re-organisation of receptors on the NK cell surface and that this rearrangement alters their effectiveness (Guia, Jaeger et al. 2011). In contrast
49 to transcriptional reprogramming, this mechanism may potentially confer NK cells with greater plasticity converting from an unresponsive state to a state where they are able to respond to stimuli, as demonstrated by NK cell adoptive transfer experiments (Elliott, Wahle et al. 2010) (Joncker, Shifrin et al. 2010).
In addition to inhibitory KIRS having an established role in NK cell education it has been proposed that interaction between a self-MHC ligand and an activating KIR may represent an additional mechanism that complements education via inhibitory receptors, such as in the case of KIR2DS1 (Fauriat, Ivarsson et al. 2010). Expression of KIR2DS1 together with its self- ligand leads to reduced NK cell responses following activation with cellular targets; a phenomenon that is best fitting with the disarming hypothesis. However the authors speculated that the resulting NK cell hyporesponsiveness could represent a parallel event to arming via inhibitory receptors. It is therefore likely, that both inhibitory and activatory receptors act along a complex continuum refining NK cell education. A more recent study implicated the NKp46 receptor as a checkpoint in NK cell regulation. This work revealed an unexpected aspect of NK cell education, suggesting that the engagement of this activating receptor can downregulate NK cell responsiveness (Narni-Mancinelli, Jaeger et al. 2012).
Whether NKp46 participates in the tuning of effector function is an interesting paradox that remains to be substantiated in humans.
Although substantial progress has been made, the work to identify the precise the mechanisms involved in NK cell stages of development, education and target recognition as a friend or foe is ongoing and clearly important for the engineering of innovative therapeutic strategies based on the manipulation of NK cell immunity.
50 NK cell arsenal of weapons and regulation - an overview
Since the original discovery of NK cells it has become increasingly apparent that their effector functions do not conform to stereotypical biological roles. NK cells have a range of weapons at their disposal and are capable of inducing cell death in target cells via contact dependent mechanisms (Vivier, Tomasello et al. 2008). Cytotoxicity is exerted via lytic granule release and perforin-mediated mechanisms or via pathways involving the engagement of death receptors (Smyth, Cretney et al. 2005) (Krzewski and Coligan 2012).
The functional importance of these pathways and their role in the pathophysiology of many diseases has being examined in gene-targeted mice lacking these molecules, and humans with genetic mutations (Smyth, Cretney et al. 2005) (Orange 2006) (Wood, Ljunggren et al.
2011). A number of surface and adhesion molecules enabling effector–target cell interaction have been implicated in NK cell target cell recognition and cytotoxicity (Yokoyama 1993)
(Moretta, Bottino et al. 2001). The best characterised of these molecules is CD16, which elicits antibody dependent cellular cytotoxicity (ADCC), following crosslinking by IgG antibodies bound onto a target cell (Perussia, Tutt et al. 1989) (Ravetch and Perussia 1989).
The complex processes directing NK cell cytotoxicity i.e. release of lytic granules and co- operation of the various signaling pathways are gradually being dissected (Krzewski and
Coligan 2012). In brief, lysis of target cells by NK cells involves the formation of an immune synapse followed by release of lytic granules in a tightly regulated process to avoid indiscriminate killing (Topham and Hewitt 2009). An integral part of this process involves actin polymerization and co-operation of signals from NK receptors such as CD16 with the integrin LFA-1, which promotes polarisation of lytic granules and directed degranulation
(March and Long 2011).
In addition to perforin-mediated mechanisms of induction of cell death, NK cells express and utilise death ligands such as FasL and TRAIL involving the engagement of death receptors
51 (e.g. Fas/CD95, TRAIL-R) on target cells inducing classical caspase-dependent apoptosis
(Smyth, Cretney et al. 2005) (Wallin, Screpanti et al. 2003). Although a key role for death receptor mediated apoptosis has been established in the selective targeting of tumours by
NK cells (Screpanti, Wallin et al. 2001), the full spectrum of the in vivo biological functions of
FasL and especially TRAIL is less well defined. Interestingly, the involvement of these two ligands in the preferential killing of target cells may depend on the maturation stage of NK cells. It has been previously reported that immature NK cells, defined as CD161+ CD56−, target cells through a TRAIL-dependent pathway, while mature NK cells, CD161+ CD56+, can utilise both FasL and TRAIL to induce target cell apoptosis (Zamai, Ahmad et al. 1998).
Exactly how this phenomenon is regulated requires a more detailed analysis of FasL and
TRAIL expression on NK cells. A number of cancer studies have supported an immunosurveilance role of TRAIL in the liver (Smyth, Cretney et al. 2001) and at immune privileged sites (Phillips, Ni et al. 1999) (Lee, Herndon et al. 2002). However, evidence is mounting that TRAIL signaling through its receptors is also involved in non-apoptotic functions i.e activation of pro-survival and proliferation signalling through NF-κB, JNK and p38 mitogen-activated protein kinase (MAPK) pathways (Kimberley and Screaton 2004)
(Aggarwal 2003). Additional roles for TRAIL have been suggested in normal haematopoiesis and innate immune responses regulation (Zauli and Secchiero 2006) (Hameed, Arnold et al.
2012) (Steinwede, Henken et al. 2012). Collectively, published evidence highlights the complexities of TRAIL and its receptor multicomponent signaling, aspects of which will be discussed further in later sections.
NK cells are multipotent lymphocytes capable of exerting functions beyond direct target cell killing. They have a prodigious ability to produce cytokines and chemokines that activate, recruit and suppress other cells of the innate and adaptive immune arm. Traditionally it was considered that CD56bright NK cells are the main cytokine producers with little capacity for
52 cytotoxicity, whereas CD56dim NK cells were proficient killers but poor at producing cytokines
(Cooper, Fehniger et al. 2001) (Fehniger, Cooper et al. 2003) (Ferlazzo, Thomas et al. 2004).
However, recent work suggested that both NK cells subsets are efficient at both cytolytic and non-cytolytic functions despite their phenotypic heterogeneity (Fauriat, Long et al.
2010). IFN-γ is the principal example of an NK cell cytokine. It is central to host resistance to many infections, regulating several genes associated with immune system functions and recruiting other effector cells to the site of infection (Boehm, Klamp et al. 1997). Its production by NK cells is known to enhance antigen presentation by antigen presenting cells
(APCs) by upregulation of MHC class I expression, potentiate macrophage killing of intracellular pathogens, shape the TH1 arm of the immune response in addition to having direct anti-proliferative effects on virus infected or transformed cells (Caligiuri 2008)
(Mocikat, Braumuller et al. 2003) (Maher, Romero-Weaver et al. 2007) (Wallach, Fellous et al. 1982). Moreover, NK cells are also a source of TNF-α, macrophage-activating factor, lymphotoxin-α (LT-α), granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage inflammatory protein (MIP), IL-5, IL-10, IL-13 and IL-3 (Caligiuri 2008) (Cooper,
Fehniger et al. 2001) (Perona-Wright, Mohrs et al. 2009) (Jinushi, Takehara et al. 2004) (Lee,
Kim et al. 2009).
As previously mentioned, the net sum of simultaneous activating and inhibitory signals determines NK cell function, which may be ultimately calibrated by local environmental factors. For instance in resting, unmanipulated NK cells the engagement of at least two different activation receptors is necessary to yield Vav1 activation sufficient to overcome inhibition mediated via the E3 ubiquitin ligase protein c-Cbl (Kim, Das et al. 2010). NK cell activation by cytokines such as IFN-α, IL-2, IL-12, IL-15, and IL-18 (Walzer, Dalod et al. 2005), however, disables the need for synergistic co-activation upon encounter of a target cell.
Instead, engagement of single activation receptors now suffices for the induction of effector
53 function (Bryceson, March et al. 2006). The key cytokines implicated in NK cell activation can be produced either by activated APCs or directly by infected cells (Nguyen, Salazar-Mather et al. 2002). Signalling through the type I IFN receptors (IFN-α/β R1 and IFN-α/β R2) enhances NK cell-mediated cytotoxicity (Garcia-Sastre and Biron 2006). This is well established in infection with MCMV and several other viruses. NK cell cytotoxicity appears to be dependent on type I IFNs, whereas IFN-γ production by NK cells requires the presence of
IL-12 and induction of secretion of IL-15 supports NK cell proliferation (Orange and Biron
1996) (Steinberg, Eisenacher et al. 2009). For instance during infection with MCMV, a robust production of IL-12 leads to potent activation of NK cells. In contrast during LCMV/HIV infection the low levels of IL-12 likely account for the weak NK cell responses (Lee, Miyagi et al. 2007). Moreover, the synergistic action of IL-12 and IL-18 primes NK cells in response to a viral attack and leads to increased translation of IFN-γ mRNA (Gherardi, Ramirez et al. 2003).
NK cell activation is induced through signaling via the janus tyrosine kinase/signal transducer and activator of transcription (JAK/STAT) pathway downstream of cytokine receptors.
Different members of the STAT family have been described to facilitate the differential effects imposed by cytokines (Garcia-Sastre and Biron 2006) (Nguyen, Cousens et al. 2000).
For example type I interferons signal via STAT1 to drive NK cell cytotoxicity and proliferation via enhanced expression of IL-15 (Nguyen, Salazar-Mather et al. 2002). On the other hand,
STAT4, and not STAT1, is necessary for IL-12-dependent production of IFN-γ by NK cells
(Nguyen, Salazar-Mather et al. 2002). Thus, STAT molecules and their regulation influences cytokine signaling and determine viral sensitivity at early time points after infection.
Moreover, the threshold for NK cell activation can also be negatively tuned by cytokines such as transforming growth factor-β (TGF-β) and IL-10 (Laouar, Sutterwala et al. 2005).
Thus, a better understanding of how environmental ‘niches’ condition the function of NK cells, is of importance in the study of NK cells in different clinical scenarios and disease contexts such as hepatotropic virus infections.
54
NK cells in antiviral defense – viral recognition and genetic studies
Evidence from both murine and human studies, and epidemiological human data, have demonstrated an important role for NK cells in the control of microbial infections, but also immunosurveillance, haematopoietic stem cell transplantation, reproduction and autoimmunity (Vivier, Tomasello et al. 2008). The molecular mechanisms whereby NK cells recognise virus-infected cells and confer resistance to viral disease remain largely unknown.
Early work in the 1980’s and subsequent studies have documented the ability of NK cells to control infection by certain viruses in experimental animal models (Welsh 1986) (Lee, Miyagi et al. 2007). Despite the fact that only few selective NK cell immunodeficiencies have been described in humans, these provide the most persuasive evidence for a role of NK cells in the defense against viral infections, in particular human herpes virus infections (Orange 2006)
(Fischer 2007) (Bryceson, Rudd et al. 2007) (Biron, Byron et al. 1989). Early after viral challenge and type I IFN production there is rapid NK cell activation and expansion, both systemically and localised in the infected area. NK cells can respond to infection, preceding an antigen-specific T-helper and CTL response, either directly by recognising infected cells and/or indirectly involving cytokines and interaction with accessory cells (Biron, Nguyen et al. 1999). Their antiviral effect is mediated via their cytotoxic activity and/or by non- cytotlytic mechanisms that may preserve the integrity of infected cells and tissues endowed with vital functions, such as the liver (Tay and Welsh 1997). Viral modification/down- modulation of MHC class I molecules and absence of appropriate inhibitory interactions in combination with expression of stress induced ligands or virus encoded ligands lead to NK cell mediated killing (Raulet 2003) (Brutkiewicz and Welsh 1995). Indirect activation via contact-dependent and soluble signals derived from accessory cells and T cells potentiate NK cell effector function (Horowitz, Stegmann et al. 2011). The production of type I IFN and IL-
55 12 by plasmacytoid dendritic cells (pDC) further enhances NK cell proliferation, secretion of
IFN-γ and cytotoxicity, whereas NK cell trafficking is co-ordinated by chemokines including
MIP-1α, IP-10 and MIG (Biron 1999) (Salazar-Mather, Orange et al. 1998) (Dalod, Hamilton et al. 2003). Both pathways of NK cell activation are likely to participate in their control of viral infections. Viral evasion strategies that specifically target NK cells, subverting both pathways of activation, support their important role in antiviral defense (Lanier 2008)
(Lodoen and Lanier 2005) (Orange, Fassett et al. 2002).
The most convincing evidence of the role of NK cells in the early defence against viruses was obtained from studies of murine cytomegalovirus (MCMV) infection, demonstrating increased susceptibility or resistance to MCMV infection following NK cell depletion or NK cell adoptive transfer respectively (Bukowski, Warner et al. 1985). Direct NK cell lysis of infected cells has been found to be clearly involved in the control of MCMV infection in mice, whereas defective NK cell IFN-γ production and cytotoxicity render mice more susceptible to MCMV (Scalzo, Corbett et al. 2007) (Bukowski, Woda et al. 1984). Interaction and binding of the NK cell activating receptor Ly49H to the MCMV m157 glycoprotein is one way NK cells selectively recognise infected cells and has been found to be sufficient for viral elimination; those strains of mice expressing Ly49H are highly resistant to MCMV infection
(Daniels, Devora et al. 2001) (Arase, Mocarski et al. 2002) (Smith, Heusel et al. 2002). The characterisation of MCMV encoded molecules and interactions with other Ly49 molecules has been extended in recent years from the study of genetically different mouse strains (Orr and Lanier 2011) (Lanier 2008) (Kielczewska, Pyzik et al. 2009) (Table 1.2). The identification of MCMV genes involved in evasion of NK cell control, including downregulation of NKG2DLs and the expression of MCMV encoded decoy ligands, including MHC class I homologs that prevent NK cell stimulation or inhibit NK cell function respectively, exemplify further the importance of NK cells (Orange, Fassett et al. 2002) (Babic, Pyzik et al. 2010) (Arapovic,
56 Lenac Rovis et al. 2009). However, in contrast to MCMV, NK cells are not as crucial in early control of certain viruses such as in the case of lymphocytic choriomenginitis virus (LCMV)
(Bukowski, Biron et al. 1983) (Welsh, Brubaker et al. 1991).
Recognition of other viruses by NK cells is less well defined. For instance, resistance to ectromelia virus has ben suggested to be mediated in part by NKG2D (Fang, Lanier et al.
2008) and by CD94-NKG2A (Fang, Orr et al. 2011). The role of NCRs, another set of important NK cell triggering receptors, in the direct recognition of viral proteins, has been implicated by some recent studies (Chisholm and Reyburn 2006) (Chisholm, Howard et al.
2007) (Fuller, Ruthel et al. 2007) (Arnon, Lev et al. 2001) (Mandelboim, Lieberman et al.
2001). In particular, interaction of NKp46 with viral hemagglutinin (HA) of influenza virus and hemagglutinin-neuraminidase of parainfluenza virus, can lead to enhanced cytotoxicity against infected targets and cytokine production by activated NK cells, suggesting a potential role of NK cells in the innate defense against these viruses (Mandelboim, Lieberman et al.
2001) (Glasner, Zurunic et al. 2012). The role of NKp46 in conferring increased resistance to influenza A virus is further supported by enhanced viral sensitivity observed in NKp46 knock- out mice (Gazit, Gruda et al. 2006). NKp46 along with DNAM-1 has been reported to be involved in the response against HCMV-infected dendritic cells (DCs) (Magri, Muntasell et al.
2011). Unlike NKp46 and NKp44, NKp30 does not bind to influenza HA (Arnon, Lev et al.
2001). Instead it has been reported to interact with pp65 of human CMV (Arnon, Achdout et al. 2005) and to mediate in part clearance of vaccinia virus (VV) (Chisholm and Reyburn
2006) (Table 1.2).
In addition to NK cell-activating receptors, NK cells express toll like receptors (TLR) that may play a role in the direct recognition of infected cells and NK cell activation. Recent work from an in vivo model of vaccinia infection demonstrated that direct TLR-2 stimulation on murine
57 NK cells is critical for their activation and function in response to vaccinia virus (Martinez,
Huang et al. 2010) (Table 1.2). The implication of other NK cell surface receptors and their ligands during viral infections remain less well defined. It has been previously suggested that
HCV envelope protein 2 binding to CD81 on NK cells inhibits their effector functions (Crotta,
Stilla et al. 2002) (Crotta, Brazzoli et al. 2010) (Tseng and Klimpel 2002); however this finding has been subsequently challenged (Yoon, Shiina et al. 2009).
NK cells utilising death ligands can induce death of virally infected cells, a mechanism which may important for immature NK cells which cannot use perforin dependent cytotoxicity
(Colucci, Caligiuri et al. 2003). With respect to regulation of TRAIL expression, upregulation by proinflammatory cytokines such as IFNs and strategies employed by viruses to manipulate TRAIL signaling provided some early indications that it may be involved in the immune response to viruses (Cummins and Badley 2009) (Sedger, Shows et al. 1999) (Sato,
Hida et al. 2001). In addition to an antiviral but also pathogenic potential of TRAIL expressing
NK cells in viral hepatitis, studies from murine models have highlighted their dual role in other infections (Ishikawa, Nakazawa et al. 2005) (Sato, Hida et al. 2001) (Diehl, Yue et al.
2004). Mice only have one full length TRAIL-R, unlike humans, and the study of TRAIL-R-/- deficient mice has allowed some additional insight into the physiological role of TRAIL.
Notably enhanced resistance against MCMV was observed in TRAIL-R-/- compared to wild type control mice, attributed to enhanced inflammatory cytokine production and increased
NK cell activation (Diehl, Yue et al. 2004), suggesting that TRAIL may also act in the negative regulation of the immune response to infection.
The vital role of the MHC-specific activating NK receptor Ly49H in the response to CMV infection in mice supports a possible involvement of certain HLA-specific receptors during viral infection in humans. Although, the Ly49 lectin-like receptors do not exist in humans,
58 structurally different KIRs function similarly and recognise peptide-loaded MHC class I molecules. Carrington and co-workers have performed a number of human genetic epidemiological studies in an attempt to correlate viral pathogenesis with the presence of certain KIRs and their MHC counterparts (Carrington and Alter 2012). Consequently, findings of certain KIR genes and HLA combinations advocate for a role of NK cells in influencing the outcome of viral infections, such as clearance of HCV and HIV disease progression in humans
(Carrington and Alter 2012) (Parham 2005) (Cheent and Khakoo 2011) (Bashirova, Thomas et al. 2011) (Table 1.2). It has been proposed that the strength of the binding between certain
KIRs and their corresponding HLA/peptide ligands could potentially determine the threshold for NK cell activation and therefore viral control (Alter, Heckerman et al. 2011) (Fadda and
Alter 2011). This is demonstrated in HCV infection, where expression of the inhibitory NK cell receptor KIR2DL3 in combination with its ligand HLA-C1 has been linked with resolution of acute HCV infection (Khakoo, Thio et al. 2004) and a superior response to Peg-IFN-
α/ribavirin treatment during chronic HCV infection (Knapp, Warshow et al. 2010) (Vidal-
Castineira, Lopez-Vazquez et al. 2010). Interestingly, the nature of the peptide bound within the HLA Class I groove can have some bearing on KIR–HLA interactions influencing NK cell activation (Fadda, Borhis et al. 2010). Recognition of virally infected cells by both NK cell activating and inhibitory KIRs has also been suggested to contribute to the control of HIV-1 infection by recent epidemiological and functional studies (Martin, Gao et al. 2002) (Martin,
Qi et al. 2007) (Carrington and O'Brien 2003) (Jennes, Verheyden et al. 2006) (Alter, Martin et al. 2007) (Bashirova, Thomas et al. 2011). Individuals co-expressing the activating KIR3DS1 allele in combination with its presumed MHC class I ligand, HLA-Bw4-80I, have a significantly slower progression toward AIDS compared with individuals that lack this composite genotype (Martin, Gao et al. 2002). On a functional level, KIR3DS1+ NK cells exhibit robust cytolytic function against HIV-infected Bw4-80I+ CD4+ T cells (Alter, Martin et al. 2007).
However, despite the above evidence, a physical interaction has yet to be confirmed
59 between KIR3DS1 and Bw4-80I. Further studies showed that inhibitory subtypes of KIR3DL1 are also associated with slower HIV disease progression (Martin, Qi et al. 2007). It has been recently proposed that KIR-associated HIV-1 sequence polymorphisms on a population level can enhance the binding of NK cell inhibitory KIRs to HIV-1-infected CD4+ T cells, reducing NK cell antiviral activity (Alter, Heckerman et al. 2011). The finding of such sequence polymorphisms at a population level may be a demonstration of how host-pathogen co- evolution has promoted the selection of viral variants with the ability to escape NK cell responses. Although an extensive and detailed analysis of the impact of KIR-HLA association in HBV infection is lacking, a susceptibility effect of the B haplotype has been suggested from the study of KIR genes in a small cohort of CHB patients compared to spontaneous resolvers and healthy controls (Lu, Zhang et al. 2008). An additional study comparing CHB patients with healthy controls identified KIR2DL3:HLA-C1 homozygosity as protective, whereas the
KIR2DL1:HLA-C2 association conferred increased susceptibility to persistent HBV infection
(Gao, Jiao et al. 2010). More recently a novel KIR-dependent function, by which a variety of infectious agents may directly stimulate NK cells has been reported, such as in the case of
KIR3DL2 mediated CpG DNA uptake and shuttling to early endosomes (Sivori, Falco et al.
2010).
Table 1.2. Evidence for antiviral role of NK cells
Viral Host Effector Function Infection /Observed effect HCMV Human NK cell deficiencies increase susceptibility to infection HSV mainly by herpes viruses (Biron, Byron et al. 1989) (Orange VZV 2006) (Orange 2006) MCMV Mouse Protective against MCMV, perforin and IFN-γ mediated (Bukowski, Biron et al. 1983) (Bukowski, Warner et al. 1985) (Welsh, Brubaker et al. 1991) (Scalzo, Fitzgerald et al. 1990) (Orange, Wang et al. 1995) Mouse Mouse Protects against mouse pox requires IFN-γ, role for NKG2D Pox virus and CD94/NKG2A (Chaudhri, Panchanathan et al. 2004) (Fang, Lanier et al. 2008) (Fang, Orr et al. 2011) Ebola Mouse Protective - perforin dependent (Warfield, Perkins et al. 2004)
60 Direct recognition of viral protein by NK cells Virus Host/Receptor Effector function MCMV Mouse Ly49H Recognises m157 (Arase, Mocarski et al. 2002) MCMV Mouse Ly49P Recognises H-2D (Desrosiers, Kielczewska et al. 2005) Influenza Mouse NCR1 Critical function in the (Gazit, Gruda et al. 2006) virus eradication of influenza Influenza Human NKp46 Recognition of viral (Mandelboim, Lieberman virus haemagglutinin et al. 2001) Vaccinia Human NCRs Increased susceptibility to lysis (Chisholm and Reyburn Virus by NK cells following recognition 2006) (Chisholm, HSV via NCRs Howard et al. 2007) MCMV NKp46 and DNAM-1 (Magri, Muntasell et al. 2011) Filovirus Human NKp30 NKp30 dependent cytotoxicity (Fuller, Ruthel et al. of virus infected dendritic cells 2007) Other Receptor Ligand interactions Virus Host/Receptor Effector function Virus Mouse/Human Recognises stress inducible (Guma, Angulo et al. infected NKG2D molecules on infected cells 2006) cells HIV Human KIR3DS1 Interaction with HLA-Bw4 , (Martin, Gao et al. 2002) Human KIR3DL1 delayed progression to AIDS (Martin, Qi et al. 2007) Distinct allelic combinations of KIR3DL1 are also associated with slower HIV disease progression HCV Human KIR2DL3, Interaction with HLA-C1, rapid (Khakoo, Thio et al. KIR3DS1 clearance 2004) EMCV Murine TRAIL TRAIL mediated cytotoxicity of (Sato, Hida et al. 2001) virally infected cells induced by IFNα/β
In recent years there is accumulating evidence showing that chronic viral infections, such as
HIV and HCV, alter the relative proportions of NK cell subsets, receptor expression and effector function (Bjorkstrom, Ljunggren et al. 2010). Although the findings on NK cell receptor expression in chronic HCV have been inconsistent (Cheent and Khakoo 2011), the redistribution of NK cell subsets and expansion of aberrant NK subpopulations during the course of these chronic viral infections, define an important aspect of pathogenesis that can modify innate immune responses. In CHB infection emphasis has been given to the potential pathogenic role of NK cells. However, their antiviral potential has been poorly investigated
61 up until recently. A more detailed account of the role of NK cells in infection with HBV will be provided in chapter 3 (antiviral control) along with an analysis of their effector function during persistent infection.
NK cell cellular crosstalk and immunoregulatory role
It has been increasingly acknowledged that NK cells exert a variety of immunoregulatory factors taking on the role of ‘conductors’ of the inflammatory response, in addition to NK cell direct antimicrobial activity. Although, NK cells rely on dendritic cells (DCs) for efficient activation they can reciprocally induce DC maturation via release of TNF-α and cell-cell contacts. However, they can also kill immature DC (iDC) in humans and mice via NKp30,
NKp46 and DNAM-1-mediated recognition, thereby influencing DC homeostasis (Spaggiari,
Carosio et al. 2001) (Pende, Castriconi et al. 2006) (Ferlazzo and Munz 2004) (Fig. 1G). This mechanism reduces the pool of iDCs during immune activation, but allows fully activated
APCs to present antigens to T cells. Hepatic NK cells have also been shown to eliminate iDC in vivo in a TRAIL dependent manner, thereby hampering the generation of T cell immunity to DC vaccination (Hayakawa, Screpanti et al. 2004). In addition to selectively editing APCs during the course of immune responses, NK cells are also capable of regulating the extent/magnitude of these responses. In support of this, activated macrophages have also been found to be susceptible to NKG2D mediated NK cell cytotoxicity (Nedvetzki, Sowinski et al. 2007) (Lunemann, Lunemann et al. 2008) and to TRAIL-mediated cytotoxicity (Steinwede,
Henken et al. 2012). Thus, through the downmodulation of other innate responses NK cells may represent a way of eliminating overstimulated macrophages, preventing the development of immunopathology (Nedvetzki, Sowinski et al. 2007). This is supported by the findings from MCMV infection of mice deficient in perforin mediated killing or depleted of
NK cells. In these cases there is accumulation of highly activated TNF-α producing
62 macrophages and severe disease, reminiscent of haemophagocytosis lymphohistiocytosis like syndromes (van Dommelen, Sumaria et al. 2006).
Besides influencing innate cell function NK cells can shape adaptive immune responses not only indirectly via promoting cross-presentation of antigens by subsets of DCs (Krebs, Barnes et al. 2009) but also by direct regulation of T and B cells. In the inflamed lymph node, NK cells can promote the priming of CD4+ T helper type 1 (TH1) cells by secreting IFN-γ
(Mailliard, Alber et al. 2005). Moreover, early NK cell mediated control of viral load during infection, may indirectly promote T cell responses by inhibiting high levels of viral replication which can have an immunosuppressive effect (Bukowski, Woda et al. 1984). There are also emerging data that activated NK cells might communicate directly with T cells via a process involving cognate cell-cell interactions. Activated human NK cells have been shown to express major histocompatibility complex (MHC) class II and, akin to mature DCs, can present antigens directly to T cells (Hanna, Gonen-Gross et al. 2004) (Hanna and
Mandelboim 2007). More recent studies by Zingoni et al indicated that activation of NK cells induces the expression of CD86 (a B7 family member) and OX40L on NK cells, the ligands for
CD28 and OX40 respectively, required for co-stimulation and optimal activation/proliferation of T cells (Zingoni, Sornasse et al. 2004) (Fig. 1G). A supportive role of NK cells in B cell activation and promotion of isotype class switching has also been reported (Yuan, Wilder et al. 1992) (Gao, Dang et al. 2001) (Yuan 2004) (Wilder, Koh et al.
1996) (Gao, Jennings et al. 2008) (Sinha, Guo et al. 2012).
In addition to shaping T cell responses in a positive fashion, NK cells can also restrain these, contributing to the resolution of adaptive immune responses and prevention of autoimmunity (Bukowski, Woda et al. 1984) (Su, Nguyen et al. 2001) (Robbins, Bessou et al.
2007) (Lee, Kim et al. 2009) (Andrews, Estcourt et al. 2010) (Stadnisky, Xie et al. 2011)
63 (Mitrovic, Arapovic et al. 2012) (Narni-Mancinelli, Jaeger et al. 2012). NK cells can mediate suppression of T cells promoting maintenance of homeostasis through the release of cytokines, such as TGF-β and IL-10, which can inhibit DC maturation or T-cell activation and functions, and/or through the direct lysis of activated T-cells similar to the elimination of
APCs (Andoniou, Coudert et al. 2008) (Lee, Kim et al. 2009; Vivier and Ugolini 2009) (Perona-
Wright, Mohrs et al. 2009) (Waggoner, Cornberg et al. 2012) (Lang, Lang et al. 2012)
(Rabinovich, Li et al. 2003) (Cerboni, Zingoni et al. 2007) (Soderquest, Walzer et al. 2011)
(Fig. 1G). NK cell lysis of T cells should come as no surprise given that T cells were identified as one of the earliest targets, including mouse YAC-1 cells and primary thymocytes, for NK cell cytotoxicity in vitro and in vivo respectively (Hansson, Kiessling et al. 1980) (Hansson,
Kiessling et al. 1979). Moreover, following activation, T cells were shown to upregulate
NKG2DLs and become susceptible to autologous NK cell mediated lysis (Rabinovich, Li et al.
2003) (Cerboni, Zingoni et al. 2007) (Roy, Barnes et al. 2008), unless the T cells expressed sufficient amounts of classical or non-classical MHC class I molecules. Consequently, blockade of CD94-NKG2A inhibitory receptors led to NK cell cytotoxicity against activated
CD4+ T cells, and amelioration of experimental autoimmune encephalomyelitis (EAE) in mice, suggesting the use of blocking antibodies to NKG2A to prevent CD4+ T cell-dependent autoimmunity (Lu, Ikizawa et al. 2007). More recently a central role for NK cell killing in mediating immunoregulatory effects has emerged during chronic viral infections. Studies from the LCMV mouse model indicate that NK cytotoxicity directed towards antiviral T cells regulates the balance between viral clearance versus persistence and disease immunopathogenesis (Waggoner, Taniguchi et al. 2010) (Waggoner, Cornberg et al. 2012)
(Lang, Lang et al. 2012) (Welsh and Waggoner 2013). NK cells may further contribute to T cell exhaustion in LCMV infection via limiting the ability of APCs to stimulate proliferation of
T cells, as an additional mechanism to the direct regulation of T cells (Cook and Whitmire
2013). The potential of NK cells to restrain/influence the efficacy of the adaptive antiviral
64 response in CHB will be considered further in chapter 4.
Type&I&IFN,&IL$12,&& IL$15,&IL$18& Ac:vated& mDC& T&cell&
Cytotoxicity&IL$10&
NK&cell& IFN$γ,&& IFN$γ,&TNF$α& ?co$s:mula:on&& Cytotoxicity&
T&cell& iDC&
Figure 1G. Interactions with adaptive and innate immune cells.
Besides their immunoregulatory effects and contrasting their protective role in various inflammatory conditions, NK cells can facilitate immunopathology directly. As previously discussed, NK cells through the destruction of infected cells may compromise the function of vital organs, as in the case of liver inflammation (Dunn, Brunetto et al. 2007). A recent study has also highlighted a deleterious role of NK cells during severe influenza infection promoting mortality of the host (Zhou, Juang et al. 2013). Another mechanism includes the release of pro-inflammatory mediators and contribution to an overwhelming systemic inflammatory response recently described in experimental sepsis (Chiche, Forel et al. 2011).
A detrimental contribution of NK cells has been also postulated in inflammatory and autoimmune conditions (Vivier, Tomasello et al. 2008). Alternatively dysregulation of NK
65 cells Ieading to massive killing of uninfected ‘bystander’ cells has been implicated in the development of AIDS (Vieillard, Strominger et al. 2005).
Thus, NK cell activity can be a double edge sword that needs to be tightly regulated in order to achieve optimal control of infectious agents and pathogen specific immunity in the absence of overwhelming damage to the host.
Emerging concept in NK cell biology. Do NK cells remember?
An exciting recent development in NK cell biology is the emerging notion that NK cells can exhibit a memory response during secondary antigenic exposure to viral infections, confering long-lasting protection (Sun, Lopez-Verges et al. 2011) (Vivier, Raulet et al. 2011).
Until recently, immunological memory was a feature attributed to the adaptive arm of the immune system. In a seminal study in mice lacking T and B cells, a subset of liver NK cells expressing CXC-chemokine receptor 6 (CXCR6) was found to mediate hapten-specific contact hypersensitivity, a prototype ‘adaptive’ immune reaction (Paust, Gill et al. 2010) (Paust and von Andrian 2011) (O'Leary, Goodarzi et al. 2006). Moreover, studies of viral infections further support the concept that NK cells may be capable of exhibiting some form of immunological memory. As an example, during MCMV infection the Ly49H+ NK, recognising the MCMV m157 protein, were reported to clonally expand and to subsequently contract
(Sun, Beilke et al. 2009). A few remaining long lived cells could be detected up to 3 months after infection and mounted a secondary response with enhanced IFN-γ secretion and cytotoxicity compared to naïve cells upon ex vivo restimulation. These ‘virus-experienced’ cells, when transferred to naïve immunocompromised mice, were shown to be protective against a lethal virus challenge. Although there is no unique memory marker, these long lived MCMV expanded NK cells expressed high levels of KLRG1, an inhibitory receptor that
66 recognizes cadherins (Sun, Beilke et al. 2009). Consistent with a ‘memory-like’ phenotype,
NK cells activated in vitro by cytokines (IL-12/IL-18) also led to the generation of NK cells displaying more robust IFN-γ production following activating receptor stimulation compared with resting NK cells (Cooper, Elliott et al. 2009). The increased frequency of IFN-γ producing NK cells among re-stimulated PBMCs is also encountered in the context of vaccination in an antigen, CD4+ T cell and IL-2 dependent manner (Horowitz, Hafalla et al.
2012). Furthermore, in acute infection with human cytomegalovirus (HCMV) there is a preferential expansion of NK cell populations expressing the activating receptor NKG2C
(Lopez-Verges, Milush et al. 2011). Equally in another longitudinal study of acute hantavirus infection in humans, there is a prolific expansion and long term persistence of NKG2C+ NK cells with greater numbers observed in HCMV seropositive individuals compared to HCMV seronegative individuals, demonstrating a virus driven clonal expansion of an NK cell subset suggestive of the existence of memory NK cells in humans (Bjorkstrom, Lindgren et al. 2011).
Interestingly, the expression of CD57 on long-lived NK cells after the contraction phase may provide a possible memory marker for human NK cells. In one study increased frequencies of
NKG2C-expressing NK cells in HBV-infected patients were reported, however the sero- prevalence for CMV within this cohort was not accounted for (Oliviero, Varchetta et al.
2009).
Taken together, these findings have prompted ongoing research into the mechanisms that allow the boosted effector function of NK cells to be generated and maintained, with the ultimate goal of targeting NK cells in vaccine strategies and improving pathogen control.
67 NK cells in context: the liver microenvironment
The unique features of NK cells in various organs may reflect their capacity to adjust to different microenvironments and assimilate tissue-specific functions, such as hypothesized for uterine NK cells during placentation (Hanna, Goldman-Wohl et al. 2006). NK cells represent about half of all human hepatic lymphocytes recently suggested to contain a
CXCR6+ population of cells with memory like properties (Norris, Collins et al. 1998) (Doherty,
Norris et al. 1999) (Paust, Gill et al. 2010). Importantly the phenotype and effector function of both resident and recruited NK cells exhibits a characteristic repertoire and cytokine profile (Lassen, Lukens et al. 2010) (Krueger, Lassen et al. 2011). This is supported by adoptive transfer experiments, where splenic NK cells migrating to the liver assume a phenotype and function closely resembling that of liver-resident NK cells (Lassen, Lukens et al. 2010). During infection with viral pathogens there is also a profound accumulation of NK cells in the liver (Salazar-Mather, Orange et al. 1998). The production of chemokines by organ specific cell types may be therefore important in the selective NK cell homing during physiological and disease conditions.
In addition to normal distribution and trafficking, tissue specific NK cell functions could be further affected by in situ differentiation secondary to local microenvironmental influences.
Notably, human liver NK cells have a CD56bright phenotype, and lack CD16 expression and therefore do not correspond to the typical mature circulating CD16+CD56low NK cells (Shi,
Ljunggren et al. 2011), raising the possibility that NK cells may also develop locally. The liver represents the main site of haematopoiesis during foetal life. Although its activity reduces dramatically during adult life, with the bone marrow becoming the predominant site, the liver maintains its capacity to support haematopoiesis, such as in the setting of BM dysfunction in adults (Golden-Mason and O'Farrelly 2002). A recent study by Moroso and
68 colleagues demonstrated that all stages of NK cell development are detected in the human adult liver, where hepatic NK cell precursors, continuously recruited from the periphery into the liver can differentiate into ‘liver-specific’ NK cells (Moroso, Famili et al. 2011).
Hepatic NK cells face the challenging task of offsetting immunity to pathogens with tolerance to food and self-antigens. Any alteration of hepatic NK cell subsets and function may therefore contribute to the defective clearance and persistence of infections especially with pathogens that have a predilection for the liver, such as HBV. Overall, the tolerogenic nature of the liver maintains intrahepatic NK cells in a functionally hyporesponsive state.
During steady state, gut derived lipoteichoic acid (LTA) and lipopolysaccharide (LPS), bind
Toll-like receptor 2(TLR-2) and TLR-4 respectively on intrahepatic Kupffer cells, triggering the release of interleukin-10 (IL-10) (Tu, Bozorgzadeh et al. 2008). In turn IL-10 blunts the response of liver NK cells to IL-12/IL-18 stimulation. Moreover, the liver contains a significant population of NK cells expressing the inhibitory receptor NKG2A, and lacking expression of the activating receptor LY49. Although, the timing and circumstances under which IL-10 is released require further investigation, IL-10 mediated induction of NKG2A in the liver may regulate resident NK cell function (Krueger, Lassen et al. 2011). A dialogue between liver resident cells and NK cells might therefore allow the fine tuning of hepatic NK cells during injury, transformation or infection (Fig. 1HA,B). In humans it has been reported that the intrahepatic compartment compared to peripheral blood comprises of a reduced number of licensed NK cells expressing inhibitory receptors for self-MHC-I (Burt, Plitas et al.
2009) (Norris, Doherty et al. 2003). In addition the relatively low or absent expression of
MHC-I by hepatocytes (Daar, Fuggle et al. 1984) (Lau, Bird et al. 1993) may further contribute to establishing functional tolerance, containing auto-aggression. However, pro- inflammatory cytokines in the inflamed liver contribute to an activated phenotype of NK cells and can overcome hyporesponsiveness (Kim, Poursine-Laurent et al. 2005) (Cooley,
69 Xiao et al. 2007) (Yokoyama and Kim 2006). In support of this, in vitro cytokine stimulation of liver NK cells results in potent effector function and cytotoxicity (Burt, Plitas et al. 2009).
Notably in vitro derived NK cells were highly functional, despite the lack of expression of
KIRs, supporting the notion that NK cell responsiveness is not a rigid but rather a dynamic phenomenon shaped by the local milieu and changes in the MCH class I microenvironment
(Moroso, Famili et al. 2011). Interestingly, during steady-state, NK cells are preferentially situated within the hepatic sinusoids, often found in close proximity to the endothelial cells
(Krueger, Lassen et al. 2011). Through the fenestrations in the sinusoidal lining or following migration in the liver parenchyma, NK cells can establish direct contact with infected hepatocytes. Thus it is plausible that intrahepatic NK cell function may be further adjusted by the balance of signals received from hepatocytes, which have the potential to upregulate cellular stress ligands (Chen, Wei et al. 2007).
Although TRAIL is not expressed on intrahepatic NK cells taken from healthy living donor transplanted tissue (Ishiyama, Ohdan et al. 2006), in the context of CHB, there is an enrichment of TRAIL found on the preferentially activated CD56Bright NK cell subset (Dunn,
Brunetto et al. 2007). TRAIL expression by human liver NK cells could be induced by IFN-α, by contrast TRAIL expression by mouse immature liver NK cells appears to be under the control of IFN-γ. One plausible explanation for these findings is that TRAIL-dependent killing by NK cells could serve as an inducible defence mechanism against infection and/or cellular transformation (Huntington, Vosshenrich et al. 2007). Moreover, activated NK cells can lyse hepatocytes or stellate cells via a TRAIL dependent pathway during HBV infection or fibrosis respectively, highlighting their potential dual function as previously discussed (Dunn,
Brunetto et al. 2007) (Taimr, Higuchi et al. 2003) (Fig. 1HB). Recently the intrahepatic accumulation of NK cell populations displaying a unique functionality has also been demonstrated in HCV infection. NKp46High expression was found to define a specific NK-cell
70 subset with a potential involvement in both the suppression of HCV replication and induction of HCV-associated liver damage reinforcing the role of NK cells in HCV immunopathogenesis (Kramer, Korner et al. 2012). The finding that a proportion of liver NK cells express NKp44 (Burt, Plitas et al. 2009) poses the question whether this subpopulation could represent activated NK cells or the IL-22-producing NKp46+ RORγt+ cells, so far described in mucosa surfaces, which have the capacity to be hepatoprotective (Zenewicz,
Yancopoulos et al. 2007). However, NKPs isolated from fresh liver perfusates showed no evidence of commitment to an (lymphoid tissue inducer) LTi-lineage or the ability to produce IL-22 (Moroso, Famili et al. 2011).
A" Steady#State#
Hepatocytes# #
Stellate#cell# Disse
Space## of# LSEC
IL610# • Increased#NKG2A# LTA# • Low#LY49# KC# NK# LPS# • Hyporesponsiveness##
Sinusoidal# lumen# #####to#IL612#and#IL618#
71 B" Viral#Infec?on# Hepatocyte#lysis#
HBV# HBV# HBV# HBV# Hepatocytes# #
Stellate# NKG2DLs Disse cell#lysis#
Space## of# LSEC
• Increased#TRAIL# • Cytotoxicity# KC# NK# Sinusoidal# lumen#
ProBinflammatory# Cytokines#
Figure 1H. Effector properties and NK cell interactions within the hepatic sinusoid during steady state (A) and viral infection (B). KC: Kupffer cell; LSEC: liver sinusoidal endothelial cell; LTA: lipoteichoic acid; LPS: lipopolysaccharide; TRAIL: TNF related apoptosis inducing ligand; NKG2DLs: NKG2D ligands.
A shortcoming of the vast majority of human studies is that analysis is often restricted to the peripheral compartment and phenotypic and functional evaluation of intrahepatic NK cells is often lacking or limited to hepatectomy specimens often obtained from cancer patients; obtaining liver samples being the limiting factor. Future studies addressing the factors involved in human hepatic NK cell development and maturation would increase our current understanding of NK cell biological functions and mechanisms involved in the functional shaping of liver NK cells. Improved insight into the organ specific nature of NK cells will allow us to exploit organ-intrinsic factors for therapeutic manipulation such as boosting specific
NK cell functions to control infection, whilst attenuating excessive activation in order to protect against immunopathology.
72 NK cells in HBV infection
Aims of the study
NK cells are important antiviral effector cells that may contribute to HBV control through the production of cytokines such as IFN-γ, however their role in CHB has remained poorly investigated. So far data from persistent HBV infection in humans have highlighted the pathogenic potential of NK cells and contribution to liver inflammation. We have postulated that in addition to contributing to hepatocyte damage, NK cells have defective effector function in CHB, leading to impaired antiviral control and dysregulation of adaptive immune responses.
Specific aims:
1. Determine whether in CHB there is a bias towards NK cell tolerance and defective
antiviral potential. To address this we will investigate the role of the
immunosuppressive cytokine environment.
2. Determine whether HBV-specific CD8+ T cells become susceptible to NK cell-
mediated killing in CHB through death receptor pathways concentrating on
TRAIL/TRAIL-R interactions.
73 2. Material and Methods
Flow cytometry has been utilised extensively, as an important tool, throughout this thesis.
Optimised multicolour immunofluorescence panels for high-resolution analysis of receptor expression in order to permit a detailed phenotypic and functional characterisation of human NK cells along with global and virus specific T cells have been developed. Functional parameters were assessed on the basis of previously described assays. The use of immunofluorescence and immunohistochemistry complemented and provided a strong visual corroboration of the flow cytometric data. This study benefited from access to a large number of intrahepatic samples and great efforts were spent towards optimising intrahepatic lymphocyte isolation.
Antiviral role
Separation of PBMC
Ficoll-Hypaque density gradient centrifugation was used to isolate PBMCs from whole blood.
Blood collected in vacutainers with EDTA-coated beads were transferred into 50ml Falcon tubes (Sarstedt), and the volume of blood was made up to 50ml using RPMI 1640 (Invitrogen
#21875-034). 25ml of blood was layered onto 20ml Ficoll-Paque PLUSTM (GE Healthcare #17-
1440-03) and centrifuged at 2000rpm for 23 mins with minimum brakes. PBMCs lying between the plasma and the Ficoll layer were collected and washed twice with RPMI. Cells were counted and used immediately or resuspended in freezing medium [FBS (Invitrogen,
#10106-169) with 10% DMSO (Sigma #D2650)] at 10 million cells per ml, for later use. Cells were frozen in cryovials (Nunc #366656) in aliquots of 5 million - 10 million cells.
Thawing cells
PBMCs retrieved from liquid nitrogen or -80°C freezer were thawed in a 37°C water bath and washed with 15ml DPBS (Invitrogen #14040-091) prior to counting and plating. Cell manipulations were done in 96-well round bottom plates (Sarstedt) and approximately
74 500,000 cells were plated in each well. For ex vivo dextramer staining of virus-specific T cells,
at least 1 million cells were plated per well.
Media
Complete RPMI (CRPMI) was made up as follows: 500ml of RPMI 1640 (Invitrogen #21875-
034) supplemented with 10% FBS (Invitrogen, #10106-169), 10ml of 100U penicillin and
100μg/ml streptomycin solution, 10ml of 20mM HEPES, 2.5ml of 0.5mM sodium pyruvate,
5ml of MEM non-essential amino acids (1x), 10ml of MEM essential amino acids (1x)
(Invitrogen) and 0.5ml mercaptoethanol.
Processing of liver samples and isolation of intrahepatic lymphocytes
Intrahepatic lymphocytes were isolated as previously described(Dunn, Brunetto et al. 2007).
Briefly, liver tissue was suspended in RPMI 1640 (Sigma) and macerated with a plunger from
a 25ml syringe and a scalpel in a Petri dish. The cell suspension was then passed several
times through a 70mm cell strainer (BD biosciences), washed three times and resuspended
in RPMI complete medium with 10% fetal bovine serum for counting. Lymphocytes were
identified under high magnification by their size, shape and granularity.
Antibodies and reagents for analysis of NK cells
Antibodies CD107a PE 555801 BD Pharmingen CD16-APC-CY7 557758 BD Bioscience CD3 PerCPCy5.5 332771 BD Bioscience CD56 FITC 345811 BD Bioscience CD56 PE 06824 BD Bioscience CD8 APC 555369 BD Bioscience CYTOPERM/CYTOFIX 554722 BD Bioscience HLA-A2 FITC MCA2090F Serotec IFN-γ APC 341117 BD Bioscience IFN-γ PE IC285P R&D Systems IL-10 APC 554707 BD Pharmingen Mouse IgG1-PE PN IM0670 Coulter Immunotech TRAIL-PE 550516 BD Pharmigen TNF-α PE 559321 BD Pharmingen
75
Reagents anti-IL-10 Receptor (CDw210) 556011 BD Pharmingen Anti-TGFβRII 241-R2-025 R&D Systems b-Mercaptoethanol 31350010 Gibco Bovine Serum Albumin A-7906 Sigma Brefeldin A B7651 Sigma CD56 MicroBeads 130-050-401 Miltenyi Biotec CFSE V12883 Molecular Probes Cytometric Bead Array (CBA) 558274 BD Biosciences Human IL-10 Flex Set (Bead B7) CBA 560383 BD Biosciences Human IL-17A Flex Set (Bead B5) CBA 560429 BD Biosciences Human TGF-β1 Single Plex Flex Set Cytoperm/Cytofix 554722 BD Biosciences DMSO D2650 Sigma EDTA E-7889 Sigma Essential amino acids 11130.036 Gibco Fetal Bovine Serum 10108-165 Invitrogen Ficoll-Paque PLUS 17-1440-03 GE Healthcare Formaldehyde F8775 Sigma Functional grade anti-IL-10 16-7108-85 eBioscience HEPES 15630056 Gibco Human IL-10 High Sensitivity Elisa kit 850.880.096 Diaclone Human IL-12 premium grade 130-096-704 Miltenyi Biotec IL-2 Human 11 011 456 001 Roche Ionomycin I0634 Sigma Monensin M5273 Sigma NK cell Isolation Kit 130-092-657 Miltenyi Biotec Non-essential amino acids 11140.035 Gibco Penicillin/streptomycin 15070.063 Gibco Phorbol 12-Myristate 13-Acetate P8139 Sigma Phosphate Buffered Saline P4417-100 TAB Sigma RPMI 1640 21875-034 Invitrogen Recombinant Human IL-18 B003-5 R&D Systems Recombinant Human IL-10 34-8109 eBioscience Saponin S-4521 Sigma Sodium pyruvate 11360.039 Gibco Trypan Blue T8154 Sigma Zenalb 20 (Human Albumin 20%) Bio Products Lab.
76 Extracellular staining and Flow Cytometric analysis
For phenotypic analysis, PBMC isolated from HBV patients and healthy donors were stained with fluorochrome-conjugated antibodies to CD3-Cy5.5/PerCP, CD56-FITC, CD16-APC Cy7, and TRAIL-PE or isotype matched controls (BD Biosciences, Cowley, U.K.). In selected experiments TRAIL expression was determined following overnight incubation with 50ng/mL of rhIL-10 (eBioscience). PBMC were acquired on a FACS Calibur flow cytometer (Becton
Dickinson) and analysed using Flowjo analysis software (Treestar).
IFN-γ production by intracellular staining
As previously described (Dunn, Peppa et al. 2009), PBMC were incubated with 50 ng/mL of rhIL-12 (Miltenyi) and rhIL-18 (R&D Systems, Abingdon, U.K.) for 21 hours at 37°C. 1mM monensin (Sigma-Aldrich, Gillingham, U.K.) was added for the final 3 hours. Cells were fixed and permeabilised followed by intracellular staining for IFN-γ-PE (R&D systems). Where indicated the same experiments were performed in the presence of rhIL-10 (50ng/mL), or blocking antibodies to IL-10 (5 µg/mL) (eBioscience) and IL-10R (10 µg/mL) alone or in combination with anti-TGF-βRII (10 μg/mL) (BD Biosciences). NK IFN-γ production was determined by subtracting baseline IFN-γ production from that observed after cytokine or antibody treatment. NK cells from PBMC of a randomly selected group of patients were isolated (>96% purity and viability) (Miltenyi Biotec, Germany, NK isolation kit) to assess the effect of exogenous IL-10 on IFN-γ production.
CD107 degranulation assay
As previously described (Dunn, Peppa et al. 2009), PBMC were incubated with K562 cells (5:1
E:T ratio) for 3 hours at 37°C following overnight stimulation with a combination of rhIL-
12/rhIL-18 or medium alone in the presence or absence of rh-IL10. CD107a-PE antibody (BD
Biosciences, Cowley, U.K.) was added at the time of stimulation with target cells and 1mM
77 monensin was added during the last two hours of the incubation prior to staining and acquisition.
Determination of Serum Cytokine concentrations by Cytometric Bead Array (CBA)
CBA flex-sets were used for the determination of IL-10, IL-17 and TGF-β (BD Biosciences,
Cowley, U.K ) according to manufacturers’ protocols for serum samples.
Regulatory role
NK cell isolation and sorting
Freshly purified NK cells from PBMC of CHB patients were isolated (>96% purity and viability)
(Miltenyi Biotec, Germany, NK isolation kit) as per the manufacturer’s instructions. NK cells were depleted by CD56 MACS microbeads (Miltenyi Biotec, Germany). Where indicated separated NK cells were plated into TWs (1µm pore size, Polycarbonated Membrane,
Corning Costar). Fluorescence activated cell sorting of NK cells (99% purities) was performed on the basis of CD56 expression (CD56+CD3-) by FACSAria (Becton Dickinson) (Fig. 2A).
Control PBMC stained with the same antibodies were passed though the machine untouched.
Figure 2A. NK cell purity.
78 Antibodies and reagents
Antibodies 7-AAD VIABILITY STAIN 00-6993-50 EBioscience ANNEXIN V FITC 556420 BD Biosciences ANNEXINV Pacific Blue 640917 Biolegend DAKO peroxidase blocking reagent S2001 DAKO Casein solution 10x SP-5020 Vector labs CD16-APC-CY7 557758 BD Biosciences CD19 Krome Orange A96418 Beckam Coulter CD27 FITC 555440 BD Biosciences CD27 V500 561223 BD Biosciences CD3 PeCY7 25-0038-42 EBioscience CD38 V500 562288 BD Biosciences CD38 FITC 555459 BD Biosciences CD4 APCeFluor780 47-0049-42 EBioscience CD45RA PE 555489 BD Biosciences CD45RA V450 48-0458-42 EBioscience CD56 FITC 345811 BD CD56 PE 06824 BD CD56 PE ECD A82943 Beckman Coulter LIVE/DEAD Fixable Dead cell stain kit 620435 Invitrogen CD57 V450 48-0577-42 EBioscience CD57 APC 560845 BD Biosciences CD8+ ALEXA Fluor700 56-0086-82 EBioscience HLA-A2 FITC MCA2090F Serotec HLADR V450 9048-9952-120 EBioscience HLADR V500 561224 BD Biosciences IL-2 FITC IC202F R&D IL-10 APC 554707 BD Pharmingen IFN-γ V450 560371 EBioscience IFN-γ APC 341117 BD Biosciences ImmPACT NovaRED SK-4805 Vector labs Mouse IgG2B Isotype PE IC0041P R&D Mouse IgG1 k isotype APC 17-4714-41 EBioscience Mouse IgG1 k Isotype PERCP 46-4714-80 EBioscience Mouse IgG1-PE PN IM0670 Coulter Immunotech PD1-PERCP 462799-42 Ebioscience TRAIL-PE 550516 BD Pharmigen TRAIL-R2 FAB6311P R&D Systems TRAIL-R1 FAB347P R&D Systems
General and immunohistochemistry/ Immunoflourescence Reagents
79 ANNEXIN V staining Buffer 422201 Biolegend anti-CD3 M725429-2 Dako anti-CD8 VP-C325 Vector labs anti-DR5 ab8416 Abcam anti-NKp46 AF1850 R&D Systems anti-mouse DyLight 488 DI-2488 Vector labs anti-rabbit DyLight 594 DI-1594-1.5 Vector labs Biotinylated anti-goat secondary BA-5000 Vector labs Carboxyfluorescein FLICA apoptosis #92 Immunohistochemistry detection kit- Polycaspases FLICA Technologies/Serotec FAM-VAD-FMK Carboxyfluorescein FLICA apoptosis #917 Immunohistochemistry detection kit- Polycaspases SR-VAD- Technologies/Serotec FMK Carboxyfluorescein FLICA apoptosis #99 Immunohistochemistry detection kit- Caspase 8 FAM-LETD- Technologies/Serotec FMK Cell staining Buffer 420201 Biolegend EnVision FLEX wash buffer K800721 DAKO Mayers haematoxylin 3801582E Leica Mouse ImmPRESS MP-7402 Vector labs Streptavidin-ABC alkaline SA-5100 Vector labs phosphatase Total Cytoxicity and apoptosis #971 Immunohistochemistry detection kit Technologies/Serotec Recombinant Human TRAIL-R1 Fc 347-DR R&D Systems Chimera Recombinant Human TRAIL-R2 Fc 631-T2 R&D Systems Chimera Recombinant Human IgG1 Fc, CF, 110-HG-100 R&D Systems Human IgG1 VectaMount H-5000 Vector labs VECTASHIELD mounting medium H-1200 Vector labs with DAPI
For phenotypic analysis, PBMC isolated from HBV patients and healthy donors were washed
in PBS, and surface stained at 4°C for 20 min with saturating concentrations of monoclonal
anti-CD3 PE-Cy7, CD8+ Alexa700, HLADR V500, CD19 V450, CD4 APC-Cy7 (eBioscience),
CD56-TEXAS Red (Beckman Coulter, High Wycombe, U.K.), and TRAIL-R2 (R&D) in the
presence of fixable live/dead stain (Invitrogen) (Fig. 2B representative example of gating
strategy). Where stated the degree of activated caspase 8 and activated pancaspases was
80 determined using the FAM-LETD-FMK or the carboxyfluorescein-(FAM-VAD-FMK) FLICA kit
(Serotec) according to the manufacturer’s instructions. For further phenotypic analysis of intrahepatic CD8+ T cells the following antibodies or isotype matched controls were used:
CD38-FITC (BD Pharmigen), CD45RA V450 (eBioscience), CD27 V500 (BD Biosciences, Oxford,
U.K.), PD1-PERCP (eBioscience), CD57-APC (BD Pharmigen) in the presence of fixable live/dead stain (Invitrogen). Where indicated the viability of CD8+ T cells was further assessed by staining for Annexin V (Biolegend) according to the manufacturer’s protocol in the presence of 7AAD viability staining solution. Cells were acquired on a LSRII (BD biosciences) and analysed using flowjo.
81 Figure 2B. Representative example of gating strategy. Lymphocytes were gated using forward and side scatter after the exclusion of doublets. Fixable live/dead stain was used to identify live cells. NK cells and T cells were identified using CD56, CD3, CD4 and CD8 markers. Gates were set according to isotype staining levels for TRAIL-R2 as shown.
Identification of virus specific CD8+ T cells
The frequencies of HBV peptide specific cells from HLA-A2 positive individuals were evaluated directly ex vivo or following short-term culture by multimer staining as previously described. Briefly total PBMCs were stained with APC-labelled HBV c18-27, envelope 183-
191, envelope 335-343, envelope 348-357 and polymerase 508-510 dextramers (Immudex,
Denmark) at 370C for 15 min in complete RPMI plus 10%FCS. The cells were then pelleted and stained as above. A control dextramer was used to identify the population of positive cells. For the analysis of HCV-specific CD8+ T cells PE-labelled HLA-A2 restricted MHC class I tetrameric complexes specific for HCV NS3 1406-1415 (KLSGLGINAV) and HCV NS3 1435-
1443 (CVNGVCWTV), were used (a kind gift from Dr E. Barnes, Oxford University). Tetramer staining was considered positive if a distinct population (>0.02%) could be discriminated.
HLA-A2 peptide sequence Type Viral protein Amino acid FLPSDFFPSV Dextramer HBV core 18-27 FLLTRILTI Dextramer HBV envelope 183-191 KLHLYSHPI Dextramer HBV polymerase 508-510 WLSLLVPFV Dextramer HBV envelope 335-343 GLSPTVWLSV Dextramer HBV envelope 348-357 NLVPMVATV Dextramer CMV pp65 495-504 KLSGLGINAV Tetramer HCV NS3 1406-1415 CVNGVCWTV Tetramer HCVNS3 1435-1443
Peptide stimulation
PBMC or PBMC depleted of NK cells were stimulated with the following peptides representing HLA-A2 restricted viral epitopes: HBV envelope epitopes: FLLTRILTI,
82 WLSLLVPFV, LLVPFVQWFV, and GLSPTVWLSV; HBV core epitope: FLPSDFFPSV; HBV polymerase epitopes: GLSRYVARL, KLHLYSHPI); CMV pp65 immunodominant epitope:
NLVPMVATV; EBV BMLF1 immunodominant epitope: GLCTLVAML and Influenza MP 58-66 immunodominant epitope: GILGFVFTL0 (Proimmune). For stimulation of HLA-A2 negative patients a pool of 15mer peptides overlapping by 10 residues spanning the core protein of
HBV genotype D (kindly provided by Christian Brander IrsiCaixa AIDS Research Institute-
HIVACAT, Barcelona, Spain). For control viral responses in HLA-A2 negative patients, CMV peptides spanning the pp65 protein were used (JPT Peptide Technologies). PBMC from HCV infected patients were stimulated with HCV peptides. Amino acid sequences of the specific antigenic HCV peptides were identical to those of the respective MHC class I tetrameric complexes used (kindly provided by Dr E. Barnes, Oxford University). Peptides were dissolved in sterile endotoxin free DMSO. Final DMSO concentration during culture was
<0.1%.
Peptide amino acid sequence HBV env 183-191 FLLTRILTI HBV env 335-343 WLSLLVPFV HBV env 338-347 LLVPFVQWFV HBV env 348-357 GLSPTVWLSV HBV pol 455-463 GLSRYVARL HBV pol 502-510 KLHLYSHPI HBV core 18-27 FLPSDFFPSV HCMV pp65 495-504 NLVPMVATV EBV BMLF-1 259-267 GLCTLVAML INFLUENZA A MP 58-66 GILGFVFTL HCV NS3 1406-1415 KLSGLGINAV HCV NS3 1435-1443 CVNGVCWTV
Short-term culture
Where indicated PBMC or PBMC depleted of NK cells were stimulated with1mM peptide (or a pool of the seven HBV peptides) in the presence of 20IU IL-2 in RPMI complete medium for
83 10 days at 37oC. IL-2 and medium were refreshed on day 4 of culture. In selected experiments NK cells were depleted from PBMC culture at 24 hours (Day 1). On day 9, PBMC were re-stimulated with 1mM peptide overnight, in the presence of Brefeldin A (added 1 hour into the incubation). In selected experiments a physiological ratio of freshly isolated NK cells was re-added in the culture either at the onset of stimulation or at day 10. Where indicated isolated NK cells were plated into TWs (1 µm pore size, Polycarbonated
Membrane, Corning Costar) at onset of culture. Virus-specific T cells were identified either via dextramer staining as previously described relative to control dextamer or via ICS for IFN-
γ after subtracting the frequency of IFN-γ detectable without any peptide stimulation. Briefly cells were surface stained, fixed and permealised followed by intracellular staining for IFN-γ
APC (BD biosciences). To examine the effect of blocking TRAIL on virus specific CD8, 1 µg/mL of TRAIL-R2/Fc (R&D) or IgG1-Fc (R&D) control antibody was added with peptide at onset of culture and cells were treated as described above. The degree of pancaspase activation was determined using the carboxyfluorescein-(FAM-VAD-FMK) FLICA apoptosis detection kit
(Serotec) according to the manufacturer’s protocol for detection by flow cytometry. FLICA reagent was added at day 10 of culture, for an hour prior to staining.
PBMC from HCV infected patients were stimulated with 1mM (pooled peptides) and expanded as above. HCV-specific T cells were identified via tetramer staining as previously described.
Overnight stimulation
For overnight stimulation of PBMC or IHL, 10mM peptide was added for 12 hours and the cells were incubated at 37oC in the presence of Brefeldin A (added 1 hour into the incubation). To examine the effect of TRAIL blocking these experiments were repeated in the presence of a combination of TRAIL-R2/Fc and TRAIL-R1/Fc chimeras (R&D) added at the time of peptide stimulation. HBV specific T cells were identified by ICS for IFN-γ.
84 Immunohistochemistry and Immunofluorescence
Sections of archival paraffin embedded HBV tissues were dewaxed, rehydrated and epitope retrieval performed as previously described (Dunn, Brunetto et al. 2007). All washes for immunostaining techniques and dilution of antibodies were with EnVision FLEX wash buffer
(Dako). Following 2 minutes in Harris haematoxylin to block auto-fluorescence, immunostaining was performed on a Shandon Sequenzer. After a 10 minute endogenous protein block in 2% Casein solution (Vector), primary antibodies mouse anti-CD8+ (Vector) at
1/50 dilution and polyclonal rabbit anti-DR5 (Abcam) at 1/100 were applied for one hour.
Visualisation was performed with anti-mouse DyLight 488 and anti-rabbit DyLight 594
(Vector) at 1/200 dilution for 15 minutes. Sections were mounted with VECTASHIELD, with
DAPI (Vector) and captured on a Zeiss Axiovision microscope with X1000 magnification.
Immunohistochemistry was performed on a Dako autostainer. Following a 10 minute peroxidase block (Dako) a 10 minute endogenous protein block in 2% Casein solution was applied. Sections were incubated with mouse anti-CD3 (Dako) at 1/100 dilution for 30 minutes, visualised with mouse ImmPRESS (Vector), 30 minutes and ImmPACT NovaRED
(Vector) for 5 minutes. After a water wash, 2% Casein solution was re-applied. Sections were then incubated in goat polyclonal anti-NKp46 (R&D) at 1/100 dilution for one hour, followed by biotinylated anti-goat secondary (Vector), strept-ABC alkaline phosphatase (Vector), as per manufacturers recommendations and visualisation completed with Vector blue for 20 minutes. After counterstaining with Meyers haematoxylin (Leica) slides were rapidly dehydrated, cleared and mounted in VectaMount (Vector). Images were captured on a Leica
DM with Nikon Coolpix camera at X1260 magnification.
Statistical analysis
Statistical significance was performed between paired samples using the Wilcoxon signed rank test and between HBV patients and healthy controls using the Mann-Whitney U test.
85 The non-parametric Spearman test was used for correlation analysis. P<0.05 was considered to be significant for all tests.
86 3. Antiviral NK cell role in chronic HBV infection
Background
NK cells represent a major component of the innate arm of the immune response and, as such, have been regarded to provide one of the initial barriers of cellular host defence against acute infection. However, when chronic infection becomes established NK cells may exhibit dysregulated effector function. NK cells are greatly enriched in the intrahepatic compartment, the site of HBV replication (Doherty, Norris et al. 1999) (Dunn, Brunetto et al.
2007), highlighting their potential contribution to the containment of infections with a predilection for the liver. Their antiviral role may be particularly important and/or compensatory in patients with CHB, in whom the virus-specific CD8+ T cell arm of protection is characteristically diminished and hyporesponsive (Maini, Boni et al. 2000; Maini and
Schurich 2010). However, very little is known about the quality of NK cell effector functions during persistent infection with HBV, which may equally disable their antiviral potential.
What is the evidence to support the hypothesis that NK cells are involved in the control of viral replication in HBV infection? Elucidation of the full spectrum of immunological requirements and direct role of NK cells in HBV clearance has been challenging, given the limitations of the available experimental models. Work on acutely infected chimpanzees initially implicated NK cells in the early control of HBV replication, where a substantial drop in viral replication occurs in the presence of intrahepatic INF-γ and TNF-α production, prior to the onset of adaptive immunity (Guidotti, Rochford et al. 1999; Thimme, Wieland et al.
2003). However, subsequent work underpinned a critical role for T cells in this model
(Thimme, Wieland et al. 2003). A role for NK cells in HBV control has also been supported by data from the transgenic mouse model (Kakimi, Guidotti et al. 2000). With the increasing power of mouse genetics it has been possible to further demonstrate an NK cell complimentary/supporting role for HBV clearance in the hydrodynamic injection model of
87 acute HBV (Yang, Althage et al. 2010). An early activation of NK cells has been shown in the woodchuck model of acute hepatitis followed by a transient but significant decrease in viral replication (Guy, Mulrooney-Cousins et al. 2008). Thus, the activity of NK cells is likely to control HBV infection in the early stages mainly via the production of cytokines with direct antiviral capacity, preceding the upregulation of HLA class I expression on hepatocytes
(Guidotti and Chisari 2001).
Only a few studies have examined the role of NK cells in early acute infection with HBV in humans, which is characterised by delayed propagation following infection and a lack of clinical manifestations of an early viral infection (Bertoletti, Maini et al. 2010). This is in marked contrast with other infection such as HIV, HCV, HCMV or dengue virus where typically a cytokine storm picture is observed (Stacey, Norris et al. 2009) (Nguyen, Salazar-
Mather et al. 2002). These observations and findings from the chimpanzee model showing a lack of induction of type I IFN genes in the liver during the entry and expansion phase of HBV
(Wieland, Thimme et al. 2004) have led to the widely held belief that HBV goes undetected by the innate cells. Challenging this concept, an early rise in circulating NK cells with faster kinetics than HBV-specific T cells and earlier peak of activity has been observed in the incubation phase of HBV infection in humans, suggesting that they may contribute to the initial containment of the virus (Webster, Reignat et al. 2000) (Fisicaro, Valdatta et al. 2009).
Longitudinal assesment of NK cell subsets in a patient cohort with acute HBV showed that
NK cells have an activated phenotype during the acute symptomatic phase displaying increased cytolytic activity and IFN-γ production, which subsequently decreases coinciding with both viral clearance and recovery from liver injury (Zhao, Li et al. 2012). Further work from an rare cohort of patients sampled in the early pre-clinical phase of HBV and followed up to resolution of infection has highlighted the role of the immunosuppressive cytokine IL-
10 in actively suppressing early NK cell IFN-γ at peak viraemia (Dunn, Peppa et al. 2009)
88 providing an opportunity for viral escape. Thus rather than being silent, HBV virus may be efficient at counter-acting the actions of these innate effectors in early infection, which may serve as an opportunity for viral escape. However, there is a paucity of available information from humans immediately after HBV inoculation regarding the involvement of NK cells before detectable viral replication. Equally, studies in the acute phase of HCV are very limited owing to the difficulty in identifying patients with true acute HCV infection, which is also frequently asymptomatic. Nonetheless current evidence suggests that hepatocytes following exposure to HCV and HBV may respond in a differential manner (Wieland and
Chisari 2005).
During established chronic infection with hepatotropic viruses, different small studies have attempted to address the impact of NK cells concentrating largely in the peripheral compartment. In the case of chronic HCV infection in particular there are controversial data on the phenotype and function of NK cells, which may be reflective of donor variation and disease heterogeneity (reviewed by (Mondelli, Varchetta et al. 2010). In the context of CHB, we have previously identified an enrichment of activated TRAIL+ NK cells in the liver, with up to 40% of total intrahepatic NK cells staining TRAIL+. Similar to the periphery, the majority of
TRAIL was found on the preferentially activated CD56bright NK cell subset, which mediated non-antigen specific liver damage through this death pathway. The CD56bright subset is a potent source of cytokines such as IFN-γ (Cooper, Fehniger et al. 2001) (Vivier, Tomasello et al. 2008), a crucial cytokine influencing adaptive immunity and the delicate balance between protective responses and deleterious effects. IFN-γ production can contribute to non- cytolytic control of HBV by enforcing cell-intrinsic antiviral defences in infected hepatocytes
(Guidotti, Ishikawa et al. 1996) (Kakimi, Guidotti et al. 2000). NK cell-derived IFN-γ could therefore represent an essential antiviral mechanism in tissues with vital functions that are characterised by an inherent relative resistance to perforin/granzyme dependent
89 cytotoxicity, such as the liver (Willberg, Ward et al. 2007) (Tay and Welsh 1997). Distinct organ-dependent mechanisms for the control of viral infections by NK cells has been previously advocated, suggesting the importance of IFN-γ production in the liver for the control of MCMV infection (Tay and Welsh 1997).
In this chapter we define a defect in IFN-γ production by peripheral NK cells in CHB (Peppa,
Micco et al. 2010), also confirmed by data from several other labs (Oliviero, Varchetta et al.
2009) (Tjwa, van Oord et al. 2011), which may be a key aspect leading to virus persistence.
The mechanisms leading to this dysfunctional cytokine production remain under- investigated. The cytokine microenvironment plays a crucial role in fine-tuning the intensity and quality of NK cell effector function. We have previously demonstrated that the action of
IFN-α, induced during flares of CHB, drives the expression of TRAIL on NK cells in line with subsequent observations in the setting therapeutic IFN-α (Dunn, Brunetto et al. 2007)
(Micco, Peppa et al. 2012). Likewise, NK cells in HCV infection can be polarised towards cytotoxicity and expression of TRAIL following exposure to both endogenous (Ahlenstiel,
Titerence et al. 2010), or therapeutic IFN-α (Stegmann, Bjorkstrom et al. 2010). Conversely, intrahepatic NK cell function can be downregulated by the immunosuppressive cytokine IL-
10 produced by Kupffer cells (Tu, Bozorgzadeh et al. 2008) or through the action of TGF-β
(Laouar, Sutterwala et al. 2005). More recently, a role for IL-17 in restricting NK cell function was established in disseminated vaccinia virus infection of mice with pre-existing dermatitis
(Kawakami, Tomimori et al. 2009). In this study we have explored cytokine-driven modulation of IFN-γ production by NK cells in patients with CHB and the potential to restore their non-cytolytic antiviral function.
90 Study Cohort
Patients and Healthy Subjects
Clinical assessment and blood sampling were performed during routine hepatitis clinics, with informed consent and local ethical board approval of the Royal Free Hospital, the Royal
London Hospital and Camden Primary Care Ethics Review Board. All patients were anti-
Hepatitis C- and anti-Human Immunodeficiency Virus-antibody negative and treatment naïve with the exception of a sub-group of 22 patients suppressed on a combination of
Lamivudine and Adefovir. Patient characteristics are included in Table 3.1. Paired peripheral blood and liver biopsy specimens (surplus to diagnostic requirements) were obtained from 8
CHB-infected patients (Table 3.2).
Table 3.1. Characteristics of study population. na =not applicable
Healthy HBV HBV Treatment Controls Patients Patients Group High ALT Low ALT (Lamivudine and n=31 n=29 n=35 Adefovir) n=22 Age, years (median 30 43.5 32 43 and range) (18-52) (23-65) (23-65) (18-70)
Sex Female/male 14:17 14:15 16:19 5:17
ALT IU/L (median and na 112 34 25 range) (57-604) 10-47 (18-70)
HBV DNA IU/mL na 1,546,000 870 <100 (median and range) (1150- (100- 2.900e+008) 3.300e+008) HBeAg+ na 18/29 3/35 6/22
91
Table 3.2. Patient characteristics with available liver biopsy specimens
Patients Age Sex HBeAg+ HBV ALT Necro- Modified n=8 Median M:F 2/6 DNA (IU/L) inflammatory ISHAK 35.5 6:2 (IU/mL) Median score Stage Range Median 56 Fibrosis 24-66 66,879 Range Range 15-113 646- 1.2x106
Pt1 25 F Pos 113,757 113 2/18 1/6
Pt2 32 M Pos 310,000 63 4/18 3/6
Pt3 49 M Neg 700,000 15 na 1/6
Pt4 40 F Neg 20,000 26 3/18 1/6
Pt5 24 M Neg 947 86 2/18 1/6
Pt6 66 M Neg 646 56 3/18 1/6
Pt7 39 M Neg 6500 26 3/18 1/6
Pt8 27 M Neg 1.2x106 56 3/18 1/6
92 Results
3.1 Expansion of CD56bright subset
To explore NK cell effector potential in the setting of persistent hepatitis B virus infection, we first analysed the frequency of CD56bright(CD16dim/neg) and CD56dim(CD16pos) NK cell subsets in peripheral blood of 64 patients with CHB compared to 31 healthy age-matched controls (Table 3.1). A trend for a lower percent of circulating NK cells in CHB was noted (Fig
3.1B). The proportion of circulating CD56bright NK cells was significantly increased in patients with CHB (representative FACS plots Fig 3.1A, summary data Fig 3.1C), irrespective of viral load, liver inflammation or eAg status.
To determine whether there was a further enrichment of this immunoregulatory CD56bright
NK cell subset at the site of viral replication, we compared the proportions in intrahepatic and circulating lymphocytes. In all eight patients with CHB from whom paired samples were available, the percent of CD56bright of total NK cells was higher in the intrahepatic compared to peripheral compartment (Fig 3.1D,E). Since NK cells make up a significantly greater proportion of intrahepatic than circulating lymphocytes in these patients (Fig 3.1F), this corresponds to a substantial enrichment of CD56bright NK cells in the liver.
93
94 Figure 3.1 NK cell frequency and altered subset distribution in the periphery and intrahepatic compartment
(A) Representative density plots gated on CD3-CD56+ PBMC and co-stained for CD56 and CD16 to identify NK cells from a healthy control and a CHB patient. (B) Frequency of circulating NK cells in CHB patients with low ALT and high ALT and healthy controls. (C) Summary data of the proportions of CD56bright subset in the periphery of CHB patients with low ALT (n=35, ALT <50IU/L, median 34) compared to high ALT (n=29, median ALT 112) and healthy controls (n=31). Results also presented according to eAg status and HBV DNA (High Viral load >20,000 IU/mL, Low Viral Load <20,000 IU/mL). (D) Density plots of NK cells from peripheral blood and intrahepatic lymphocytes from a representative CHB patient. (E) Paired cumulative results of peripheral and intrahepatic CD56bright NK cells frequencies from 8 patients with CHB. (F) NK cell frequency in peripheral blood and intrahepatic compartment from 8 patients with CHB with paired samples. The non-parametric Mann-Whitney U test was used to compare data between groups and the Wilcoxon signed rank test was used between paired variables. *p<0.05 or ** p<0.01 designates values that differ significantly between groups. Ctr = healthy controls.
95 3.2 Impaired non-cytolytic antiviral potential of NK cells in CHB
We have previously demonstrated that the CD56bright subset of NK cells can induce hepatocyte apoptosis through upregulation of the death ligand TRAIL during flares of eAg- negative CHB (Dunn, Brunetto et al. 2007). In this cohort of patients we confirmed an increase in TRAIL expression (largely on the CD56bright subset, Fig 3.2A representative plots) in patients with either eAg+ or eAg- CHB who had evidence of liver inflammation (Fig 3.2A summary data).
The CD56bright subset of NK cells can also be a potent source of IFN-γ (Cooper, Fehniger et al.
2001), a cytokine that has direct non-cytolytic antiviral effects on HBV replication (Guidotti,
Ishikawa et al. 1996) (Kakimi, Guidotti et al. 2000) and can promote adaptive immune responses (Vivier, Tomasello et al. 2008). Despite the enrichment of CD56bright NK cells in
CHB, we found that they had an impaired capacity to produce IFN-γ (representative plots,
Fig 3.2B). There was a significant reduction in production of IFN-γ by NK cells from 46 patients with CHB compared to 29 healthy controls (Fig 3.2B). This reduction was seen irrespective of disease activity (liver inflammation Fig 3.2B, viral load or eAg status, or method of NK cell stimulation using a combination of cytokines such as IL12/IL18, IL12/IL15 or K562 cells (Fig 3.2C)).
To assess NK cell cytolytic potential, we determined their capacity to degranulate as evidenced by CD107 expression following stimulation with K562 target cells and cytokines.
There was no significant difference in NK cell degranulation potential in 33 patients with
CHB compared to 21 controls (Fig 3.2D). Differential analysis by NK cell subset or by patient disease status did not show any differences (data not shown). NK cells in CHB were
96 therefore biased towards cytolytic and death-ligand mediated effector functions and defective IFN-γ production.
97
Figure 3.2: Higher TRAIL expression and reduced IFN-γ production by NK cells in CHB
Panels A-B and D Representative density plots from a healthy control and HBV patients with low ALT (ALT <50 IU/L, median 33) and raised ALT (ALT>50 IU/L, median 112) and summary data for TRAIL expression, IFN-γ production and CD107 expression. (C) Summary dot plots depicting NK cell IFN-γ production within the same CHB patients according to eAg status and HBV DNA (Hi and Lo VL) compared to healthy controls and summary bar charts from a subgroup of n=10 CHB patients and n=10 healthy controls comparing NK cell IFN- γ production following overnight stimulation with either IL-12/IL-18, IL12/IL-15 or IL-12/IL-18 and K562 cells (5:1 ratio) added overnight.
98
Both the CD56bright subset and the CD56dim subset (that has been recognised to also make a contribution to cytokine production (Fauriat, Long et al. 2010)) showed significantly impaired
IFN-γ production (Fig 3.2.1A). Similarly, CD56bright and CD56dim NK cells in CHB showed a trend to produce less TNF-α, despite the strong stimulus required to reliably elicit this cytokine (Fig 3.2.1B). Simultaneous assessment of IFN-γ and TNF-α production showed a significant reduction in dual producing NK cells in CHB (Fig 3.2.1C).
99
Figure 3.2.1. Defective IFN-γ production by both NK subsets in CHB
Summary bar charts comparing production of (A) IFN-γ following stimulation with a combination of IL-12/IL-18 and (B) TNF-α following stimulation with PMA/I from NK total and NK cell subsets in healthy controls and CHB patients. (C) 10 healthy controls and 12 CHB patients were evaluated for the co-production of TNF-α and IFN-γ following stimulation with PMA/I. Summary bar charts show the percentage of total NK cells that are single positive for IFN-γ, TNF-α and double positive for IFN-γ/TNF-α. *P<.05, **P<.01, ***P<.001 by Mann- Whitney test.
100 To determine the potential of potent antiviral treatment to correct this bias in NK cell effector function, we studied a group of 22 patients with HBV viraemia well-suppressed on a combination of Lamivudine and Adefovir. Upon viral suppression and normalisation of liver inflammatory markers, there was no significant change in the percent of NK cells (Fig
3.2.2A), but the proportion of CD56bright NK cells decreased to levels observed in healthy controls (Fig 3.2.2B); in line with this, NK cell TRAIL expression reduced to baseline levels (Fig
3.2.2C). However NK cell IFN-γ production was only partially augmented upon antiviral treatment (mainly CD56dim subset, Fig 3.2.2D) and remained significantly lower than that in healthy controls (Fig 3.2.2E).
101
Figure 3.2.2 Skewed NK cell effector function in CHB is only partially corrected during therapy
(A) Frequencies of circulating NK cells in healthy controls (n=31), CHB patients (n=64) and HBV patients on antiviral treatment (Rx, n=22). Summary bar charts of CD56bright proportions, NK cell TRAIL expression and NK cell IFN-γ production from healthy, CHB and patients on antiviral therapy (B-D). (E) Summary bar chart comparing production of IFN-γ from NK total and NK cell subsets in healthy controls (n=29), CHB patients (n=46) and HBV patients on antiviral treatment (n=20). Results are expressed as mean ± SEM. Rx = treated patients. *P<.05, **P<.01, ***P<.001 by Mann-Whitney test.
102 3.3 IL-10 is induced in CHB and recapitulates the NK cell defect in IFN-γ production
Effector function of NK cells is tightly regulated by the cytokine milieu and their production of IFN-γ can be inhibited by immunosuppressive cytokines such as IL-10 (Tu, Bozorgzadeh et al. 2008; Dunn, Peppa et al. 2009) and IL-17 (Kawakami, Tomimori et al. 2009). The levels of
IL-17A were not elevated in sera from patients with CHB compared to controls (Fig 3.3A). In contrast, circulating concentrations of IL-10 were significantly increased in patients with active HBV disease (Fig 3.3A,B by CBA, confirmed by ELISA, data not shown), correlating with viral load (r=0.48, p=0.002) and ALT (r=0.37, p=0.03). IL-10 levels showed a trend to decrease on antiviral treatment but remained significantly higher than in controls (Fig 3.3C), consistent with the limited restoration of NK cell IFN-γ production in these patients.
To test whether IL-10 could induce the defect in NK cell IFN-γ production seen in CHB, we re- assessed NK cell effector function with or without the addition of exogenous IL-10. IL-10 significantly suppressed NK-cell derived IFN-γ (Fig 3.3D), particularly in those patients in whom it was not already substantially reduced (Fig 3.3E). By contrast, IL-10 had no effect on cytolytic ability or TRAIL phenotype (Fig 3.3F). Notably, the ability of IFN-α to further enhance NK cell TRAIL expression in vitro (Dunn, Brunetto et al. 2007) was not abrogated by
IL-10 (Fig 3.3G). We further confirmed that IL-10 did not affect the percent of NK cells (Fig
3.3.1A). The impact of IL-10 on IFN-γ production was consistent but more modest on purified
NK cells (Fig 3.3.1B), suggesting that some of its suppressive activity on NK cells is mediated indirectly via other constituents such as APCs. The contrasting effects of IL-10 on TRAIL and
IFN-γ expression represented differential regulation of these effector functions in the same
NK cells rather than the emergence of two distinct subsets. The small population of TRAIL- expressing NK cells present in healthy donors were at least as able to produce IFN-γ as the rest of the NK cell population (Fig 3.3.1C). The addition of exogenous IL-10 suppressed IFN-γ
103 in NK cells regardless of their TRAIL expression (Fig 3.3.1C). In line with this, gating on the expanded population of TRAIL-expressing NK cells found in CHB demonstrated that their
IFN-γ-producing capacity was no more reduced than that of the non-TRAIL-expressing fraction (Fig 3.3.1D).
104
Figure 3.3 Higher levels of circulating IL-10 in CHB
Levels of cytokines IL-17A (A) and IL-10 (B) determined using Cytometric Bead Arrays flex sets from 13 healthy controls, 14 low ALT (median ALT 35, all eAg-) and 21 high ALT patients (median ALT 115, 13eAg-). Cumulative IL-10 results including therapy group (n=13, median ALT 25) (C). Representative density plots of the effect of exogenous IL-10 on IFN-γ production by NK cells from a CHB patient (D) and paired cumulative results from 19 CHB patients (E). Summary bar charts of the effect of exogenous IL-10 on the expression of TRAIL and CD107 in 5 CHB patients (F) Results are paired and expressed as mean ± SEM. (G)
105 Summary data of the effect of exogenous IFN-α and IL-10 on NK cell TRAIL expression from n=3 healthy controls and n=3 CHB patients.
106
Figure 3.3.1 Effect of exogenous IL-10
(A) Representative FACS plots showing the effect of exogenous IL-10 on NK cells frequencies (boxed CD56+CD3-) (B) NK cells from 4 eAg- CHB patients (median ALT 50, median VL 2300) were negatively purified (>96% purities) and stimulated with IL-12/IL-18 in the presence or absence of exogenous IL-10. The effect of IL-10 is shown for the CD56bright subset (**P<.01 significance determined by paired t test). (C) Representative density plots and histograms from a healthy control and (D) a CHB patient showing NK cell IFN-γ production, gated on the CD56+CD3-TRAIL- and CD56+CD3-TRAIL+ populations, following stimulation with IL12/IL18 +/- IL-10. NK cell IFN-γ production is expressed as MFI.
107
3.4 Restoration of NK cell IFN-γ production upon blockade of immunosuppressive cytokines
Since IL-10 was induced in CHB and exogenous IL-10 was able to mimic the selective suppression of NK cell effector function, we next investigated the potential to restore NK cell
IFN-γ production by IL-10 blockade. Addition of anti-IL10/IL10-R blocking mAbs restored the ability of both CD56bright and CD56dim NK cells from patients with active CHB to produce IFN-γ
(mean 2.5 fold increase, Fig 3.4A,B,D). The majority of patients without biochemical evidence of liver inflammation (and with low viral loads) did not respond to this strategy (Fig
3.4C,D), in line with their lower levels of circulating IL-10 (Fig 3.3B). A subset of those patients failing to respond to IL-10 blockade did show recovery of NK cell IFN-γ production following blockade of both IL-10 and TGF-β, another immunosuppressive cytokine known to be able to inhibit NK cell production (Fig 3.4E,F).
108
Figure 3.4 IL-10 blockade alone or in combination with TGF-βRII blocking restores NK cell IFN-γ production
(A) Representative density plot from a CHB patient of peripheral NK cell IFN-γ production in the presence of anti-IL-10 and anti-IL10 receptor blocking mAb. (B) Paired summary data from CHB patients with either active disease (High ALT median 104, n=13) or (C) inactive disease (Low ALT median 33, n=9). (D) Fold change in IFN-γ produced by total NK cells following IL-10 blockade in both groups of patients. (E, F) Representative density dot plots from a CHB patient and summary bar chart of paired results from 11 patients (n=11 median ALT 42) of NK cell IFN-γ production following IL-10 blockade alone or in combination with
109 anti-TGF-βRII blocking antibodies. Stimulus = IL-12 + IL-18. Significance determined by the Mann-Whitney test for comparison between groups and the Wilcoxon signed rank test for paired data, *p<0.05, **p<0.01.
110 To investigate whether the suppression of NK cell IFN-γ was maintained at the site of HBV replication, paired liver and blood samples from eight patients with CHB were examined
(Table 3.2). CD56bright NK cell IFN-γ production showed a trend to be even lower in the liver than the periphery of patients with CHB (Fig 3.4.1A). Levels of intrahepatic NK cell IFN-γ production did not significantly correlate with levels of ALT (Fig 3.4.1B), viral load or liver histology in this small sample of patients, only one of whom had histological evidence of significant liver inflammation (Table 3.2).
111
Figure 3.4.1 Trend towards lower IFN-γ production by the CD56bright NK cell subset in the HBV infected liver
(A) Production of IFN-γ by circulating and intrahepatic NK cells and NK cell subsets from 8 CHB patients with available liver samples. Paired summary bar charts expressed as mean ± SEM. (B) Lack of significant correlation between intrahepatic NK cell IFN-γ production and ALT. Spearman statistical test was performed (r = -0.39, p=0.32).
112
Due to limited cell numbers, individual cytokine blockade could not be performed but dual
IL-10/TGF-βRII blockade reconstituted the proportion of NK cells able to produce IFN-γ
(%positive, Fig 3.4.2A) and increased their level of IFN-γ production (MFI, Fig 3.4.2B). The fold increase in the capacity of CD56bright NK cells to secrete IFN-γ upon IL-10/TGF-βRII blockade was greater in the liver than the periphery (Fig 3.4.2A,B).
113
Figure 3.4.2 Blockade of IL-10/TGF-β enhances intrahepatic NK cell IFN-γ production
(A) Representative density plots and (B) histograms for total intrahepatic NK cell and CD56bright subset IFN-γ production upon blockade with anti-lL-10, anti-IL10 receptor and anti- TGF-βRII blocking antibodies. Paired summary bar charts of fold change increase in the percentage and mean fluorescence intensity (MFI) of NK total and CD56bright IFN-γ+ cells in the periphery and intrahepatic compartment of 7 CHB (median ALT 56). Results are expressed as mean ± SEM. Stimulus = IL12 + IL18. *p<0.05 by Wilcoxon signed rank test.
114 Discussion
The contribution of NK cells in the battle against intracellular pathogens is well established
(Vivier, Tomasello et al. 2008). Increasingly, NK cells have been recognised to possess properties previously exclusively attributed to the adaptive arm, including the capacity to develop memory and tolerance (Vivier, Raulet et al. 2011). The ample numbers of activated
NK cells in the HBV-infected liver may play a role in early viral containment. IFN-γ is one of the most prominent cytokines released by NK cells and a potent non-cytolytic mechanism of viral clearance from the HBV infected liver (Guidotti, Rochford et al. 1999). However, during persistent infection, NK cells, similar to T cells, are subjected to the tolerising effects of hepatic cytokine milieu and may develop functional deficiencies limiting their antiviral capacity. In this study we demonstrate that in the setting of chronic infection with HBV and inflammation, NK cells can acquire selective defects in antiviral function, reminiscent of the graded loss of effector function displayed by exhausted T cells (Wherry, Blattman et al.
2003).
Excessive antigenic stimulation has been shown to contribute to T cell dysfunction during chronic viral infections. Mirroring these defects, functional impairment of NK cells has been described secondary to continuous interaction of the NK cell activating receptor NKG2D with its ligands, resulting in its down-modulation and NK cell tolerance (Coudert, Zimmer et al.
2005) (Oppenheim, Roberts et al. 2005). An increase in the ligands for NKG2D has also recently been demonstrated in hepatocytes replicating HBV in the transgenic mouse model
(Chen, Wei et al. 2007) suggesting that this may be a plausible mechanism in CHB. However, recent findings and our unpublished observations showed no down-regulation of NKG2D that could account for the NK cell impairment seen in CHB (Oliviero, Varchetta et al. 2009).
Akin to T cell exhaustion in CHB, upregulation of the co-inhibitory molecule Tim-3 has also
115 been reported to mediate NK cell suppression during chronic infection (Ju, Hou et al. 2010).
However, our data point to the immunosuppressive cytokine environment mediating the selective NK cell functional defects seen in this infection.
Our results from a large cohort of CHB patients on NK cell effector potential indicated conserved cytolytic capacity coupled by an increase in TRAIL-bearing CD56bright NK cells.
Despite the expansion in the subset of NK cells that is characteristically the most potent source of cytokines (Cooper, Fehniger et al. 2001), we observed a reduction in their ability to produce IFN-γ. This divergence of effector function is consistent with recent findings, demonstrating that the routes for cytokine release are through separate pathways from cytotoxic granules in NK cells (Reefman, Kay et al. 2010). In line with these distinct trafficking pathways, the release of cytokines and cytotoxic granules in NK cells is controlled via different signalling pathways (Caraux, Kim et al. 2006) (Kim, Saudemont et al. 2007).
Cytokine secretion is a complex process and the intracellular pathways available for release are often exclusively adapted to the individual cell type and cytokine (Lacy and Stow 2011).
Unique signalling events at the molecular level are beginning to be identified in determining
NK cell cytokine and chemokine generation rather than cytotoxicity (Guo, Samarakoon et al.
2008) (Malarkannan, Regunathan et al. 2007). Thus the presence of different regulatory pathways could allow for the single functional alterations observed in CHB patients. In chronic HCV infection divergent NK cell function, characterised by preserved or increased cytotoxicity and reduced cytokine production appears to be linked to altered IFN-α signaling.
From a mechanistic point of view increased signal transducer and activator of transcription 1
(STAT1) phosphorylation, was shown to skew NK cells toward cytotoxicity, and a concurrent reduction in IFN-α induced STAT4 phosphorylation produced low IFN-γ mRNA levels (Miyagi,
Lee et al. 2010) (Edlich, Ahlenstiel et al. 2012). These recent observations could account for the failure to control HCV in the liver and may partly explain the variability of responses to
116 IFN-α-based therapies. Whether these findings extend to human CHB await further confirmation.
Our results are consistent with published work supporting a functional dichotomy with conserved NK cytolytic function and impaired IFN-γ in peripheral blood from adult patients with CHB (Oliviero, Varchetta et al. 2009) (Tjwa, van Oord et al. 2011). In one study assessing intrahepatic NK cell function during CHB, NK cells accumulated in the livers of patients with active disease, in which they were activated and hypercytolytic. A concomitant increase in
IFN-γ production, in comparison to healthy subjects, was not observed. This effect depended on an imbalanced cytokine milieu and correlated with necro-inflammation during chronic
HBV infection, further supporting the idea that NK cells are predominately polarised towards cytolytic function (Zhang, Zhang et al. 2011). Further studies on the function of intrahepatic
NK cells in CHB are required to corroborate these findings and to better delineate the role of this population. In contrast, new findings from Mondelli’s group assessing the intrahepatic function of NK cells during chronic HCV infection revealed impaired degranulation compared to healthy controls and conserved IFN-γ production secondary to NKG2D-L stimulation.
Interestingly intahepatic NK cells exhibited a unique phenotype with increased levels of expression of NKG2D and NKp46 and low levels of TRAIL (Varchetta, Mele et al. 2012).
However these results are at odds with previous functional findings obtained from peripheral blood NK cells published by the same group and others (Oliviero, Varchetta et al.
2009) (Ahlenstiel, Titerence et al. 2010) (Nattermann, Feldmann et al. 2006) (Meier, Owen et al. 2005). Although this discrepancy may be partly explained by the different protocols used for stimulation and potentially the heteregenous nature of the HCV cohort studied, it highlights the limitations of human studies with hepatotropic viruses and the relative lack of functional assessment of intrahepatic NK cells relative to healthy controls using standardised assays.
117 The liver is an immunotolerant environment with a constitutive suppression of immune responses against gut derived bacterial antigens. This suppression may be mediated by IL-10 production; down-modulation of intrahepatic NK cell IFN-γ production has been associated with the localised release of IL-10 by Kupffer cells (Tu, Bozorgzadeh et al. 2008) (Lassen,
Lukens et al. 2010). IL-10 has been shown to lead to specific impairment of NK cell IFN-γ production (Tripp, Wolf et al. 1993), in contrast with IL-17 and continuous NKG2D signalling, both of which result in universal down-regulation of NK cell effector functions (Kawakami,
Tomimori et al. 2009) (Oppenheim, Roberts et al. 2005). Further evidence from murine models suggested that IL-10 can also contribute to the regulation of liver NK cells, in part, by maintaining a greater percentage of the hyporesponsive intrahepatic NKG2A+Ly49− NK
(Lassen, Lukens et al. 2010). Recently, antiviral treatment was found to improve NK cell activation, IFN-γ production and partially restored NK cell phenotype, namely downregulation of NKG2A and KIR2DL3 in peripheral blood of CHB patients (Tjwa, van Oord et al. 2011) (Li, Wei et al. 2012) (Lv, Jin et al. 2012). Moreover blocking of the interaction between NKG2A and HLA-E was reported to enhance NK cell cytotoxicity in vitro from CHB patients with active disease. Blockade of Qa-1 in HBV carrier mice encouraged viral clearance in an NK dependent fashion further supporting a role for NKG2A (Li, Wei et al.
2012). Although it was postulated that IL-10 production from hepatic regulatory CD4+CD25+
T cells may upregulate NKG2A on NK cells (Li, Wei et al. 2012) a direct link between IL-10 and
NKG2A expression has not been established in human CHB.
Our results showed that exposure of NK cells to IL-10 in vitro recapitulated the selective reduction in IFN-γ production noted in patients with CHB. Furthermore, its blockade was able to reconstitute the capacity of NK cells from patients with active HBV infection to produce IFN-γ. Interestingly, IL-10 was not able to inhibit cytotoxic degranulation and could not overcome the capacity of IFN-α to induce TRAIL, in line with the maintenance of these
118 pathogenic functions of NK cells in CHB. Although IL-10 levels were consistently modestly elevated in the serum of patients with CHB, it is likely that higher concentrations are released at the site of infection in the liver. Some of the effects of IL-10 are potentially indirect and mediated via APCs. The importance of reciprocal interaction of NK cells with other innate lymphocytes was recently highlighted by a study where HBV modulation of the pDC-NK cell crosstalk was found to contribute to HBV persistence, diminishing NK cell IFN-γ production without affecting cytotoxicity (Shi, Tjwa et al. 2012). Along these lines, during chronic HCV infection, cells of myeloid origin can mediate inhibition of NK cell effector function following interaction of the HCV non-structural protein 5A with TLR-4 on monocytes leading to a switch from IL-12 to IL-10 production and further triggering of TGF-β secretion (Sene, Levasseur et al. 2010).
A remaining question not addressed by this study is the potential source of IL-10. NK cells themselves can produce IL-10 to allow auto-suppression (Maroof, Beattie et al. 2008) and regulation of adaptive immune responses as seen in the context of HCV and MCMV (De
Maria, Fogli et al. 2007) (Lee, Kim et al. 2009). In the HBV-infected liver the cellular sources of IL-10 include a number of other candidates, including the recently described Breg populations (Das, Ellis et al. 2012). A complex regulatory network is likely to be involved in sustaining its production, as recently reported in HIV infection (Brockman, Kwon et al. 2009).
We recently reported a transient induction of IL-10 in early acute HBV infection that was temporally associated with the increase in viraemia and production of viral antigens (Dunn,
Peppa et al. 2009). Secretory proteins of HBV (HBsAg and HBeAg) may therefore interact and modulate the inflammatory environment of the liver through the induction of immunosuppressive cytokines. In our cohort of patients with CHB it was difficult to distinguish the influence of viraemia or liver inflammation, since both were increased in patients with elevated levels of IL-10. Future study of a group of patients with high viral load
119 but normal ALT (immunotolerant phase) could help to dissect the role of these factors. The fact that NK cell IFN-γ production and IL-10 levels were not significantly normalised by potent antiviral therapy suggests that the continued secretion of high levels of HBV proteins in these treated patients may play a role. In patients with low level CHB without evidence of liver inflammation, IL-10 was not elevated and its blockade alone could not rescue NK function, which instead required additional TGF-β blockade. TGF-β is another immunosuppressive cytokine that characterises the tolerising liver environment and has been shown to be increased in CHB (Flisiak, Al-Kadasi et al. 2004). TGF-β has been shown to be another key regulator of the capacity of human NK cells to produce IFN-γ, suppressing
IFN-γ and T-bet via Smad2/3/4 (Yu, Wei et al. 2006). Interestingly, impaired function of NK cells from immunotolerant CHB patients has also been attributed to the capacity of TGF-β to downregulate the NKG2D/DAP10 and 2B4/SAP pathways (Sun, Fu et al. 2012). Thus functional NK cell defects may be more pronounced in selected patient groups with CHB. A comprehensive study of NK cells during these distinct but often fluctuating phases of persistent HBV will improve our undertanding of their role during the dynamic course of infection. Race and gender related variations should also be accounted and controlled for during future studies, given the newly described association of NKp46 with these parameters and differential immunity to HCV (Golden-Mason, Stone et al. 2012). This may also reconcile some of the NK phenotypic and functional differences observed in CHB and chronic HCV infection (Mondelli, Varchetta et al. 2010)
Although the collective action of TGF-β and IL-10 may represent an important feedback mechanism preventing exuberant immune responses and tissue immunopathology, in the context of chronic infections elevated levels may lead to a generalised attenuation of innate and adaptive immune responses, preventing resolution of infection. A role for IL-10 in chronic viral infection has been provided recently by two independent studies in LCMV
120 infection, which have both shown that blockade of the IL-10 receptor is associated with resolution of infection (Ejrnaes, Filippi et al. 2006) (Brooks, Trifilo et al. 2006) (Blackburn and
Wherry 2007). These findings have been mirrored in human studies of HIV (Clerici, Wynn et al. 1994) and HCV infection (Rigopoulou, Abbott et al. 2005). Genetic studies have also highlighted the importance of IL-10 in chronic HBV infection, with polymorphisms of the IL-
10 promoter being associated with elevated IL-10 production, viral persistence and increased disease severity and progression (Cheong, Cho et al. 2006) (Miyazoe, Hamasaki et al. 2002). This association is also seen in infection with another hepatotropic virus, HCV
(Knapp, Hennig et al. 2003).
Our data suggest that immunosupressive cytokines may induce divergent effects on NK cells in CHB, having no effect on their expression of death ligands and cytolytic granules but blunting IFN-γ production. A polarised cytolytic action and NK cells expressing death ligands like TRAIL would only be able to elicit an antiviral effect in a direct one to one manner and at the detriment of hepatocyte integrity. The amelioration in liver inflammation seen on antiviral treatment is compatible with the decrease in TRAIL-expressing CD56bright NK cells that we noted in this setting. However, potent antiviral therapy was not sufficient to significantly restore NK cell IFN-γ production. NK cells would, therefore, retain an impaired capacity for non-cytolytic clearance of HBV from hepatocytes and boosting of adaptive immune responses. Although, blockade of IL-10 in vivo in rodent models has shown promising results in terms of disease resolution, translation of this finding into the clinic should be approached with extreme vigilance. IL-10 likely governs the critical balance between ameriolating immunopathology and impeding pathogen clearance. Thus blockade of IL-10 may boost anti-viral immune responses, at the expense of shifting the balance towards a pro-inflammatory state. Equally treatment with TGF-β blocking agents long term can take the break of the normal limits on NK cell cytokine production leading to a state of
121 continuous activation. The success of IL-10/TGF-β targeted therapies depends on identifying the correct time-point of drug administration and patient group who would be most responsive to as a short-term therapeutic approach.
122 4. Regulatory NK cell role and involvement of death receptors in the modulation of T cell responses in CHB
Background
T cell responses are tightly controlled to maintain immune homeostasis and limit damage to vital organs. The pressure to protect from infection/sustain antiviral defence must be balanced against the pressure to protect from immune responses, when infections become chronic. T cells in the liver, specifically, are subjected to vigorous tolerising mechanisms to prevent over-zealous responses causing tissue injury. However, these may be inadvertently exploited by hepatotropic pathogens to undermine antiviral immunity in favour of persistence (Protzer, Maini et al. 2012). Recent studies have described a role for Bim- mediated apoptosis (Lopes, Kellam et al. 2008) and T cell signalling defects (Das, Hoare et al.
2008) compounding the propensity of HBV-specific T cells to PD-1, CTLA-4 and Tim-3 directed exhaustion (Boni, Fisicaro et al. 2007) (Schurich, Khanna et al. 2011) (Nebbia, Peppa et al. 2012) in the liver, perpetuating viral infection (Protzer, Maini et al. 2012). In addition to these mechanisms, suppression or deletion by an extrinsic population may contribute to the profound T cell attrition characterising CHB. The potential for NK cells, in particular, to regulate T cell immunity has not been previously explored in human viral infections.
NK cells are important in the early direct defence against viral invasion (Orange, Fassett et al. 2002; Khakoo, Thio et al. 2004; Lodoen and Lanier 2006; Alter, Heckerman et al. 2011)
(Vivier, Tomasello et al. 2008). Besides their direct anti-viral function, NK cells encompass several mechanisms for mediating positive feed-forward effects on T cell responses to infections. As described in previous sections, NK cells promote TH1 differentiation of CD4+ T cells by IFN-γ production (Scharton and Scott 1993); restrict virus replication during MCMV infection, sustaining the availability of conventional dendritic cells to enhance adaptive T cell antiviral immunity (Robbins, Bessou et al. 2007) (Stadnisky, Xie et al. 2011) and induce
123 robust T cell responses through killing of infected cells (Krebs, Barnes et al. 2009).
Conversely, accumulating data underline the capacity of NK cells to also exert a negative regulatory effect on T cells and restrain the antiviral T cell response (Su, Nguyen et al. 2001).
This effect is achieved through restricting antigen presentation (Andrews, Estcourt et al.
2010) (Mitrovic, Arapovic et al. 2012), production of IL-10 (Lee, Kim et al. 2009) or through direct killing of T cells. In the latter case, a number of receptor–ligand interactions between
NK cells and T cells have been described to lead to autologous lysis of activated T cells
(Rabinovich, Li et al. 2003; Cerboni, Zingoni et al. 2007; Lu, Ikizawa et al. 2007; Soderquest,
Walzer et al. 2011). More recently, NK cells have been shown to play a crucial role in influencing viral clearance and immunopathology in association with LCMV infection, via direct lysis of T cells (Waggoner, Taniguchi et al. 2010; Lang, Lang et al. 2012; Waggoner,
Cornberg et al. 2012).
In this study we sought to investigate the impact of NK cells on antiviral T cell responses in the setting of persistent infection with the human hepatotropic virus, HBV. Although NK cells in patients with CHB have impaired non-cytolytic antiviral function, we have previously shown that they preserve their cytotoxic potential and upregulate the death ligand TRAIL, particularly in the intrahepatic compartment (Dunn, Brunetto et al. 2007; Peppa, Micco et al.
2010). In addition to mediating hepatocyte apoptosis, this enriched population of activated
NK cells could employ the same death ligands to delete dysregulated HBV-specific T cells in the liver (Maini, Boni et al. 2000; Boni, Fisicaro et al. 2007). The unique hepatic architecture with the extensive sinusoidal network encouraging prolonged close contact between activated NK cells and infiltrating T cells may further amplify this pathway of deletion involving TRAIL/TRAIL-R interactions.
124 Members of the TNF and TNF-R superfamily have been shown to contribute to the regulation of T cell homeostasis by induction of apoptosis (Falschlehner, Schaefer et al.
2009) (Green 2003). Apoptotic cell death plays a vital/indispensible role in the maintenance of tolerance and a balanced T cell repertoire by ensuring the removal of auto-reactive T cells as well as appropriate contraction of the immune response (Stibbe and Gerlich 1983)
(Krammer, Arnold et al. 2007). The process of apoptosis can be triggered by both the death receptor pathway (extrinsic pathway), but also the intrinsic (mitochondrial) pathway, which are closely interlinked (Green 2003). Both pathways, despite distinct means of initiation, converge downstream and involve activation of caspases and commitment to cell death
(Stibbe and Gerlich 1983). The initiator caspase 8 of the extrinsic pathway can also mediate cleavage of Bid, the pro-apoptotic Bcl-2 family member, and thereby initiate activation of the mitochondrial pathway leading to an amplification loop of apoptosis (Stibbe and Gerlich
1983) (Fig. 4A). A critical event in the intrinsic pathway is mitochondrial membrane depolarisation and release of cytochrome C and activation of caspase 9 by the adaptor protein apoptotic protease-activating factor 1 (Apaf-1), formulating the apoptosome (Hamel,
Rohrlich et al. 2003). The pro-apoptotic Bcl-2 family member Bim on mitochondrial membranes is a crucial molecule required for thymic selection (Bouillet, Purton et al. 2002) and the down-modulation of CD8+ T cell responses following superantigen stimulation or after an acute viral infection when there is limited provision of growth factors (Bouillet and
O'Reilly 2009) (Hildeman, Zhu et al. 2002) (Pellegrini, Belz et al. 2003). We have previously demonstrated the relevance of Bim in CHB infection, mediating an early attrition of HBV- specific T cells (Lopes, Kellam et al. 2008), a situation that parallels Bim mediated loss of immunodominant LCMV specific CD8+ T cells (Grayson, Weant et al. 2006). This apoptotic propensity may be a more prominent feature of liver lymphocytes. Interestingly, some early observations gave rise to the idea that the liver is the site of preferential accumulation of activated CD8+ T cells, a high proportion of which is disposed of by apoptosis (Janssen,
125 Sanzenbacher et al. 2000) (Lee, Bach et al. 2005). More recent evidence, however, has added new dimensions to the original ‘graveyard’ model, improving our understanding of the role of the liver in maintaining a balance between tolerance and effective immunity
(Hildeman, Zhu et al. 2002) (Bertolino, Bowen et al. 2007) (Bowen, McCaughan et al. 2005)
(Crispe 2009). Overall, given the tolerogenic nature of the intrahepatic environment that may bias towards deletion or anergy of responding CD8+ T cells, death by apoptosis may constitute an important pathway that during persistent infection with HBV may be hijacked by the virus to downregulate the burst of effective antiviral immunity, favouring persistence.
In this work we probed the involvement of the death receptor pathway in mediating T cell death, with special emphasis on TRAIL, which has been shown to mediate hepatocyte death during disease flares of CHB (Dunn, Brunetto et al. 2007).
TRAIL$ EXTRINSIC$$ PATHWAY$$ TRAIL)R2$
INTRINSIC$$ DNA$damage$ PATHWAY$$ TCR$signalling$ FADD$
Pro)Casp$8$ Bcl)2$ c)FLIP$ tBid$ Bid$ Bax$ Bak$ Casp$8$ Smac/DIABLO$ Bim$ Mitochondrion$
XIAP$
CytoC$ Casp$3$ Casp$9$ ATP$Apaf)1$
Apoptosome$
APOPTOSIS$$
Figure 4A. The extrinsic and intrinsic pathways of apoptosis. Engagement of death receptors by their cognate ligands in this case TRAIL-R2 by TRAIL, prompts the recruitment of different adaptor proteins and the initiator caspase 8 resulting in its autoactivation. C-FLIP regulates the activation of procaspase 8. Active caspase 8 triggers a signaling cascade that results in activation of the effector
126 caspases (i.e caspase 3) either directly or by engaging the mitochondrial death pathway mediated by the cleavage of the BH3-only protein Bid. The cleaved form of BID (tBID) is active and can bind to pro- apoptotic BAX and BAK, resulting in mitochondrial membrane permeabilisation and release of cytochrome c and Smac/DIABLO. Combination of Cytochrome c, apoptotic protease-activating factor 1 (APAF1) and caspase 9 with ATP form a functional apoptosome that results in cleavage and activation of caspase 9, which can then cleave caspase 3. DIABLO counteracts the caspase-inhibitory activities of IAPs such as X linked inhibitor of apoptosis protein (XIAP) thereby allowing full activation of caspases leading to cell death.
Published work concluded so far that TRAIL and its receptors have pleiotropic functions, extending beyond immunosurveillance against tumours and metastasis and clearance of viral infections (Benedict and Ware 2012) (Corazza, Brumatti et al. 2004) (Falschlehner,
Schaefer et al. 2009). TRAIL can bind to five different receptors so far identified: TRAIL-R1
(DR4) and TRAIL-R2 (DR5) which contain a cytoplasmic region referred to as the death domains (DD) required for activation of the extrinsic pathway of apoptosis; TRAIL-R3 (LIT,
DcR1) and TRAIL-R4 (TRUNDD, DcR2) which are cell bound, and circulating osteoprotegenerin (OPG) that lack functional DDs and are thought to be involved in the negative regulation of apoptosis or in transmitting pro-survival signals (LeBlanc and
Ashkenazi 2003) (Kimberley and Screaton 2004). The binding affinities of the TRAIL receptors for its ligand differ, with TRAIL-R2 reported to have the highest affinity, however depending on the cell type either one of the apoptosis inducing receptors can be dominant in death signaling irrespective of levels of their surface expression (MacFarlane, Inoue et al. 2005).
The initial binding of TRAIL to its cognate receptors TRAIL-R1 or TRAIL-R2 is followed by receptor trimerisation and recruitment of adaptor proteins to the intracellular domains of the receptors and assembly of the death-inducing signaling complex (DISC). Fas-associated death domain (FADD), an adaptor molecule, translocates to the DISC where it interacts directly with the death domain (DD) of the receptors (Sprick, Weigand et al. 2000). Through its respective death effector domain (DED) it recruits in turn pro-caspases 8 and 10 to the
127 DISC leading to their activation and triggering of apoptosis directly or indirectly, engaging the mitochondrial intrinsic pathway via the protein Bid (Fig 4B see also Fig. 4A). Caspase-10 is expendable for activation of apoptosis (Sprick, Rieser et al. 2002). Although TRAIL has strong apoptosis inducing properties it is increasingly recognised that signaling via its receptors, similar to other TNF family members, may be involved in non-apoptotic functions including survival and proliferation signaling (Park, Schickel et al. 2005) (Fig. 4B).
Cell(( TRAIL&R1( TRAIL&R2( TRAIL&R3( TRAIL&R4( OPG( Membrane( DR4( DR5( DcR1( DcR2( TR1(
FADD( DISC( Pro&( Casp(8( TRADD( TRAF2/RIP1(
IKK(complex( JNK( PI3K( Ac;ve( Casp(8( NF&κB( c&Jun( Akt(
Caspase(cascade( Gene(transcrip;on( Apoptosis( Survival(
Figure 4B. TRAIL receptors and signaling. Schematic representation of TRAIL signaling leading to apoptosis. In addition TRAIL, via the recruitment of a second set of adaptor proteins, TNF receptor type 1-associated death domain protein (TRADD), TRAF2 and RIP, leads to the activation of additional signaling pathways such as the phosphoinositide 3-kinase (PI3K)–Akt, nuclear factor B (NFB) and Jun N-terminal kinase (JNK) pathways.
Regulation of TRAIL-induced death occurs at different levels through the pathway, from the receptor level, to the proximal level by c-FLIP or further downstream by caspases or bcl-2
128 family members or inhibitors of apoptosis proteins (IAPs) (Oh, Perera et al. 2008) (Marsden,
O'Connor et al. 2002) (Fig 4A). At the level of the receptor, TRAIL-R3 and TRAIL-R4 have been proposed to act as decoy receptors competing for TRAIL binding (Sheridan, Marsters et al.
1997). However, whereas TRAIL-R3 may act as a competitor for TRAIL binding, TRAIL-R4 acts in a regulatory manner involving the formation of heterologous complexes with TRAIL-R2
(Clancy, Mruk et al. 2005). The degree of regulation of caspase 8 by c-FLIP is another determinant of sensitivity to TRAIL. In addition to inhibiting activation of apoptosis recent work has revealed that c-FLIP isoforms are linked with pro-survival signaling (Budd, Yeh et al.
2006) (Sacks and Bevan 2008).
Not surprisingly, the quest for the true function of TRAIL under physiological conditions has been subject to controversy. Its crucial role in autoimmunity has been inferred by studies suggesting that TRAIL may be involved in negative selection of thymocytes. An essential role of TRAIL for thymic selection was proposed, showing increased propensity to collagen induced arthritis and autoimmune diabetes in TRAIL knock out mice (Lamhamedi-Cherradi,
Zheng et al. 2003) (Corazza, Brumatti et al. 2004). However, earlier work suggested that death of activated thymocytes is independent of TRAIL (Schug, Gonzalvez et al. 2011) in agreement with a further study by Cretney et al. whereby use of different model systems for negative selection failed to confirm an involvement of the TRAIL pathway (Cretney, Uldrich et al. 2003). The use of TRAIL-R knock out mice has additionally reported no defects in thymic selection (Diehl, Yue et al. 2004). It has also been proposed that TRAIL plays a role in primary B cell and memory B cell deletion (Ursini-Siegel, Zhang et al. 2002) (van
Grevenynghe, Cubas et al. 2011) and the regulation of erythropoiesis, even though no alterations in blood haematology were reported in TRAIL-/- mice (Sedger, Glaccum et al.
2002).
Moreover TRAIL and CD95L have been implicated in controlling the T helper 1 (TH1)/T helper
129 2 (TH2) balance. Interestingly TH1 cells appear more sensitive to TRAIL-induced apoptosis in comparison to TH2 cells, which may be partly explained by higher levels of FLICE inhibitory protein (c-FLIP) inhibiting TRAIL signalling in TH2 cells following anti-CD3 stimulation (Zhang,
Zhang et al. 2003). In addition to CD95/CD95L mediating activation induced death of over- activated T cells, the participation of the TRAIL pathway has been also implicated in the termination of T cell responses by an early study (Dhein, Walczak et al. 1995) (Rieux-Laucat,
Le Deist et al. 2003). Follow-up work further suggested an increased susceptibility of CD8+ T cell blasts compared to CD4+ T blasts to TRAIL regulation (Martinez-Lorenzo, Alava et al.
1998) (Bosque, Pardo et al. 2005). A pivotal role for the TRAIL-TRAIL-R pathway in the homeostasis of a so termed ‘helpless’ CD8+ T cell subset was shown in a study by Janssen et al. (Janssen, Droin et al. 2005). Their findings suggested that in the absence of CD4+ T cells during CD8+ T cell priming the latter are unable to expand following re-challenge with their cognate antigen and are eliminated in a TRAIL dependent fashion. These findings may be of relevance within the tolerogenic liver environment where defective CD8+T cell priming without concomitant CD4+ T cell activation may favour the development of this helpless phenotype leading to long-term dysfunction (Crispe 2009). It has been recently demonstrated that this genetic programme imposed on helpless CD8+ T cells can be bypassed by the common γc cytokines (Wolkers, Bensinger et al. 2011) (Oh, Perera et al.
2008) (Barker, Gladstone et al. 2010). By comparing the transcriptional profiles of helped versus helpless CD8+ T cells a transcriptional corepressor, NGFI-A binding protein 2 nab-2 was found to be selectively upregulated in helped CD8+ T cells after re-stimulation (Wolkers,
Gerlach et al. 2012). However, a variety of genes rather than a single one is likely to be involved in sensitivity to TRAIL and TRAIL-R mediated death. However, this contingency for
TRAIL mediated apoptosis has not been confirmed by other studies examining the effect of
CD4+ T cell deficiency on CD8+ T cell responses (Sacks and Bevan 2008) (Badovinac,
Messingham et al. 2006).
130 So far available studies have unravelled a number of interesting, divergent and in some cases controversial roles for the TRAIL pathway in the immune system. In order to better appreciate the importance of this receptor-ligand interaction it is necessary to identify and study the specific targets and effectors within a particular disease context, which may dictate permissiveness to this pathway of deletion. Herein, we approach it from the angle of
NK cells, the predominant TRAIL expressing lymphocyte in the HBV infected liver, and their potential role in T cell regulation. Our data demonstrate that HBV-specific T cells upregulate a death receptor for TRAIL and become vulnerable to NK cell-mediated killing, thereby contributing to the collapse of antiviral immunity in CHB.
Patients and healthy controls
Patients were recruited from Mortimer Market Clinic (London), the Royal Free Hospital
(London), and the Royal London Hospital. Full ethical approval was obtained and each patient gave written informed consent. All CHB patients were anti-Hepatitis C- and anti-
Human Immunodeficiency Virus-antibody negative and treatment naïve. Seven patients who had resolved previous HBV infection and 18 age-matched healthy volunteers donated blood for the study. Patient characteristics are included in Table 4.1. Liver samples were obtained from 21 HLA-A2- patients with CHB and paired peripheral and intrahepatic samples, and 5
HLA-A2+ and 4 HLA-A2- patients with CHB with available liver tissue undergoing diagnostic liver biopsies (Table 4.2). Characteristics of patients with HCV and controls without viral hepatitis from whom only liver tissue was available are outlined in Table 4.3. HCV specific
CD8+ T cell responses measurable by multimer staining from 8 HLA-A2+ individuals with only available PBMC were included in the study (Table 4.4)
131 Table 4.1. Characteristics of patients from whom PBMC alone were available.
Age, Sex HBeAg+ HBV DNA IU/mL ALT
years: Female:M Median (range) IU/L: Median
Median ale (range)
(range)
CHB patients 34 4:23 3/27 1500 38 n= 27 (23-51) (36-7.3x107) (17-378)
Resolved HBV 39 1:6 NA NA NA n=7 (29-69)
Healthy 32 6:12 NA NA NA
Controls (21-49) n=18
132
Table 4.2. Characteristics of CHB patients with available paired PBMC and liver biopsy specimens.
Patients Age Sex HBeAg+ HBV DNA ALT (IU/L) Necro- Modified n=21 Median: F:M 6/21 (IU/mL) Median: 46 inflammatory ISHAK
HLAA2- 38 4:17 Median: Range: score Stage
Range: 7503 (20-180) Fibrosis
(28-54) Range: (36-
5.19X108 )
PT1 37 M - 36 180 1/18 0
PT2 53 M - 2831 34 1/18 0
PT3 40 M - 243818 47 2/18 1
PT4 35 M - 2048 46 3/18 1
PT5 42 M + 447888 47 4/18 1
PT6 31 F + 3.24X108 37 4/18 1
PT7 44 M - 6970 45 4/18 1
PT8 32 M - 2742 82 4/18 0
PT9 43 F + 5.19X108 20 1/18 1
PT10 40 M - 448 40 2/18 0
PT11 38 F + 8.36X107 71 5/18 3
PT12 33 M - 68602 76 4/18 1
PT13 54 M - 2300 20 1/18 0
PT14 32 M - 331132 32 2/18 1
PT15 32 M + 1.07X107 87 3/18 1
PT16 38 M - 89 34 1/18 0
PT17 40 M - 616 27 2/18 2
PT18 28 F + 3.98X107 32 5/18 2
133 PT19 28 M - 60 90 5/18 3
PT20 48 M - 7503 60 2/18 0
PT21 45 M - 5652257 46 5/18 1
HBV Age Sex HBeAg+ HBV DNA ALT (IU/L) Necro- Modified
Patients Median: F:M 2/9 (IU/mL) Median: 28 inflammatory ISHAK with liver 34 7:2 Median: Range:18- score Stage biopsies Range: 6583 55 Fibrosis only 22-42 Range:
*HLA-A2+ 463- n=5 9.11X108
PT22 22 F + 9.11X108 55 6/18 5
PT23 22 F - 4100 30 3/18 1
PT24 40 F - 9397 18 3/18 0
PT25 29 F + 2040391 26 3/18 0
PT26 42 F - 463 28 3/18 3
**HLA-A2- n=4
PT27 35 M - 38144 28 2/18 1
PT28 31 F - 631 43 4/18 2
PT 29 40 F - 6583 27 NA 3
PT30 34 M - 796 28 1/18 1
*5 HLA-A2+ HBV patients with available liver biopsies were used for the identification of virus specific
CD8+ T cells with multimers- no peripheral blood was available.
** 4 HLA-A2- HBV with only available liver biopsies were used for further phenotypic analysis of intrahepatic cells. NA=not available.
134 Table 4.3. Patient characteristics with available liver biopsy specimens used as controls. No PBMCs were available. Control Biopsies Age Sex HCV RNA IU/mL ALT (IU/L) Necro- Modified
inflammatory ISHAK Stage
score Fibrosis
HCV 29 F 17508 56 4/18 2
Genotype 3a
HCV 35 M 599506 380 2/18 1
Genotype 1b
HCV 29 M 2300000 35 4/18 2
HCV 53 F 1300000 327 4/18 1
Genotype 1a
HCV 34 M NA NA NA NA
HCV 36 F 198441 50 3/18 1
Genotype 3a
HCV 26 M 200000 140 5/18 2
Genotype 3a
*NAFLD 49 M Not applicable 57 NA NA
**EXPLANTS NA NA Not applicable NA NA NA n=7
NA=not available
• *Non-alcoholic fatty liver disease (NAFLD). Histology report: Steatohepatitis with moderate
steatosis (35%) and significant perivenular fibrosis
135 • **intrahepatic lymphocytes were isolated from n=7 explants from patients with no
laboratory evidence of viral hepatitis undergoing resections for liver metastases; specimens
were derived from healthy areas surrounding the tumours.
Table 4.4. HCV patient characteristics with available PBMC. Patients Age Sex HCV RNA IU/mL ALT (IU/L) Necro- Modified n=8 Median: F:M Median: Median: 51 inflammatory ISHAK Stage
49.5 1:7 1.159e+006 Range: (29- score Fibrosis
Range : Range: (44966 - 180)
(22-59) 6.947e+006)
HCV 46 F 1141156 29 4/18 3
Genotype 3a
HCV 53 M 172065 180 NA NA
Genotype 1a
HCV 51 M 6477644 42 4/18 3
Genotype 1a
HCV 33 M 44966 77 2/18 0
Genotype 3a
HCV 22 M 2674206 58 3/18 0
Genotype 1b
HCV 59 M 6947070 44 4/18 1
Genotype 1a
HCV 48 M 1176210 77 6/18 3
Genotype 1b
HCV 51 M 220000 29 NA NA
NA= not available
136 Results
4.1 Depletion of NK cells rescues virus specific CD8+ T cells
To examine whether NK cells have the potential to regulate virus-specific CD8+ T cells we initially ascertained the impact of total NK cell depletion on the magnitude of HBV-specific T cell responses. CD8+ T cells responses against a pool of peptides representing well-described
HLA-A2 restricted HBV epitopes or overlapping peptides (15 mers) spanning the core protein of HBV were identified by IFN-γ production after short-term culture. Shown in Fig. 4.1A is a representative example of HBV responses from a patient with active CHB in the presence or absence of NK cells. Stimulation of whole PBMC resulted in the characteristic low frequency responses, in line with the well-established paucity of detectable HBV-specific T cells in CHB
(Maini, Boni et al. 2000; Boni, Fisicaro et al. 2007). Upon NK cell depletion, there was an enhancement of HBV-specific CD8+ T cells, which returned to baseline levels following re- addition of purified NK cells at a physiological ratio at the start of culture. Individual responses and summary data are depicted in Fig. 4.1B, C, showing a significant recovery of
HBV-specific CD8+ T cells upon NK cell depletion from patients with CHB. To exclude any potential contribution of other lymphocyte subsets including NKT cells, depletion experiments were also performed following flow-cytometric sorting of NK cells to 99% purity
(Fig. 4.1D). Removal of NK cells also promoted the expansion of a population of CD8+ T cells able to bind HLA-A2/HBV peptide multimers (Fig. 4.1E). This implied that NK cells were influencing the number of HBV-specific CD8+ T cells surviving in culture, not just the function of pre-existing populations.
137
Figure 4.1. Recovery of HBV-specific CD8+ T cells following depletion of NK cells
(A) Representative FACS plots from a CHB patient. HBV-specific CD8+ T cells were identified by intracellular cytokine staining for IFN-γ following ten day stimulation with a pool of HBV peptides of PBMC or PBMC depleted of NK cells (ΔNK). Where indicated, a physiological ratio of NK cells was re-added in the culture at day 0 prior to stimulation. (B) Individual responses of (n=27) CHB patients and (C) matched summary data. (D) NK cells were sorted to 99% purity by flow cytometry. Control PBMC stained with the same antibodies were
138 passed though the machine untouched. Summary bar chart of (n=3) experiments. (E) HBV- specific CD8+ T cells were identified by MHC peptide multimer staining following short-term peptide stimulation in the absence or presence of NK cells or NK cell re-addition at day 0.
Representative FACS plots from a CHB patient and summary data from (n=9) CHB patients.
Bars represent the mean±SEM. ND=not detected. *** P < 0.001, **P < 0.01.
139 4.2 Differential effects of NK cells according to T cell specificity
To explore whether NK cell depletion could similarly enhance other virus-specific responses, we analysed CD8+ T cells directed against immunodominant HLA-A2 restricted epitopes from CMV, EBV and influenza in our cohort of CHB patients. In 22 patients with CHB we were able to simultaneously compare the effect of NK depletion on HBV and CMV-specific responses; those directed against an immunodominant CMV epitope showed a non- significant trend to decrease, contrasting with the increase in T cells directed against HBV in the same patients (Figure 4.2A). Similarly, EBV and influenza specific T cell responses in patients with CHB were not augmented upon the removal of NK cells (Figure 4.2B). In patients infected chronically with another hepatotropic virus, hepatitis C virus (HCV) (Table
4.4), there was also no consistent increase in T cells able to recognise 2 immunodominant
HLA-A2 restricted HCV epitopes upon NK cell depletion (Figure 4.2C). These data therefore point to differential NK cell regulation of T cells according to their antigen specificity.
140
Figure 4.2. Differential regulation by NK cells according to T cell specificity
(A) Representative FACS plots for CMV-specific responses from a CHB patient upon PBMC stimulation and in the absence of NK cells (ΔNK) and summary bar charts comparing CD8+ T cell responses upon HBV or CMV peptide stimulation within the same (n=22) CHB patients
+/- NK cell depletion. (B) Representative FACS plots for EBV and Influenza virus-specific responses from a CHB patient upon PBMC stimulation and in the absence of NK cells (ΔNK)
141 and summary data for EBV (n=6) and Influenza (n=7). (C) Example of HCV-virus specific CD8+
T cell identification by tetramer staining during short term culture in the presence and absence of NK cells and summary bar chart from n=8 HCV patients. Bars represent the mean±SEM. **P < 0.01.
142 4.3 NK cells limit HBV-specific CD8+ responses in a contact dependent manner by inducing apoptosis
Our initial findings suggested NK cells could be suppressing HBV-specific CD8+ T cells in culture or could be actively deleting them. To investigate the latter possibility, freshly purified NK cells were re-added at day 10, just six hours prior to analysis of HBV-specific
CD8+ T cell numbers. A diminished HBV-specific CD8+ T cell response was observed irrespective of the timing of NK cell re-addition (representative example and summary data
Fig. 4.3A). The speed of this effect precluded a requirement for the inhibition of proliferative expansion of virus-specific CD8+ T cells in culture, instead indicating that these populations were amenable to rapid functional inactivation or deletion by NK cells.
To distinguish between these two possibilities, HBV-specific CD8+ T cells were identified by
HLA-A2/peptide multimer rather than IFN-γ staining, after removal of NK cells at day 0 or day 1 and re-addition of a physiological ratio of freshly isolated NK cells on day 0 or day 10.
Multimer-binding CD8+ T cells were only increased if NK cells were removed at the beginning of the culture (not on day 1), and were decreased again after re-addition of NK cells on Day 0 or 10 (Figure 4.3B). These results indicated that NK cells were able to rapidly deplete the number of virus-specific CD8+ T cells surviving in culture.
The mechanism of action of NK cells on virus-specific T cell survival during short-term culture was further investigated by transwell experiments. A physiological ratio of NK cells was re- added at day 0 directly to the culture or to transwells separated from the T cells by a semipermeable membrane. When NK cells were not in contact with T cells, T cell survival was enhanced to a similar degree to that seen upon NK cell depletion (Fig. 4.3C top panel).
The degree of pancaspase activation in HBV-specific T cells (indicating apoptosis induction) was reduced when NK cells were depleted or not in contact with T cells, whereas re-addition
143 of NK cells increased the amount of early (FLICA+7AAD-) and late (FLICA+7AAD+) apoptotic events (Fig. 4.3C, D, E). Thus NK cell induction of T cell apoptosis required cell-cell contact.
Taken together, these results indicate that at least some of the impact of NK cells on HBV- specific T cells is mediated by a direct effect on their survival that is contact-dependent and results in caspase activation.
144
145
Figure 4.3. NK cells limit the survival of CD8+ HBV-specific T cells in a contact dependent manner by inducing apoptosis
(A) Representative FACS plots from a CHB patient. HBV-specific CD8+ T cells were identified by intracellular cytokine staining for IFN-γ following ten day stimulation with a pool of HBV peptides upon NK cell depletion (ΔNK) or re-addition of freshly purified NK cells
(physiological ratio) at day 0 or day 10. Summary data from (n=4) CHB patients. (B) FACS plots from a CHB patient depicting HBV-specific CD8+ T cells identified by multimer staining, following depletion of NK cells at Day 0, and from PBMC culture at 24 hours (Day 1) and re- addition of freshly purified NK cells at a physiological ratio on Day 0 and Day 10. Summary bar charts of (n=5) experiments. (C) Top panel depicts representative FACS plots from a CHB patient. HBV-specific CD8+ T cells were identified by multimer staining following short-term peptide stimulation in the absence (ΔNK) or presence of NK cells. NK cells, where indicated, were re-added at a physiological ratio directly in the culture or were plated into transwells to prevent contact. Shown in the bottom panel are the corresponding proportions of apoptotic virus-specific cells. The degree of pancaspase activation was determined by flow cytometry using the carboxyfluorescein-FLICA apoptosis detection kit. Histograms represent early apoptotic (FLICA+7AAD-) and late apoptotic (FLICA+7AAD+) virus-specific CD8+ T cells
(D) Summary stacked bars of (n=3) experiments. (E) Gating strategy for analysis of early apoptotic (FLICA+7AAD-) and late apoptotic (FLICA+7AAD+) virus-specific CD8+ T cells from experiment shown in Fig. 3C.
146 4.4 Increased TRAIL-R2 expression on T cells in CHB
A number of ligand/receptor interactions could be responsible for mediating NK cell killing of
T cells through caspase induction. We observed an increase in expression of activated caspase 8 in HBV-specific CD8+ T cell directly ex vivo (Figure 4.4.A, B). This pointed towards a death ligand receptor pathway from the TNF superfamily able to induce apoptosis through caspase 8 (Sprick, Weigand et al. 2000). We focused on the TRAIL pathway since we have previously found TRAIL to be upregulated on NK cells during episodes of HBV-related liver inflammation (Dunn, Brunetto et al. 2007; Peppa, Micco et al. 2010). We therefore investigated whether the dysregulated T cell response in patients with CHB might be because T cells are susceptible to TRAIL-mediated killing by upregulation of TRAIL death receptors. TRAIL binding to the death receptors TRAIL-R1 and TRAIL-R2, which contain intracytoplasmic domains, triggers caspase activation and induction of apoptosis (Schneider,
Thome et al. 1997; Kimberley and Screaton 2004). TRAIL-R2 is upregulated on the surface of hepatocytes during active flares of HBV infection (Dunn, Brunetto et al. 2007) and is known to have higher affinity for membrane bound TRAIL (Schneider, Holler et al. 1998; Ichikawa,
Liu et al. 2001). We therefore compared the expression of TRAIL-R2 on global T cells in CHB and healthy controls directly ex vivo. Levels of TRAIL-R2 were low on peripheral CD8+ T cells in CHB but were significantly elevated compared to the negligible levels seen in healthy controls (Fig. 4.4C). No significant correlation was observed between the levels of expression of TRAIL-R2 and any virological or clinical parameters in the periphery (data not shown).
TRAIL-R2 expression was then compared on CD8+ T cells directed against CMV or HBV within the same CHB patients, identified directly ex vivo by MHC/peptide multimer staining (Fig.
4.4D). A significantly higher proportion of HBV-specific CD8+ T cells expressed TRAIL-R2 than
CMV-specific responses (Fig. 4.4E), in line with their differential susceptibility to NK cell modulation. No upregulation of TRAIL-R2 was observed on HBV-specific CD8+ T cells
147 obtained from individuals who had previously resolved HBV infection (Fig. 4.4F, G). These data indicated that TRAIL-R2 was preferentially induced on HBV-specific CD8+ T cells encountering their cognate antigen.
148
149
150 Figure 4.4. Higher levels of TRAIL-R2 on T cells in patients with CHB
(A) The degree of activated caspase 8 was determined directly ex vivo in peripheral HBV and
CMV specific CD8+ T cells within the same CHB patients. The results demonstrate 7AAD-
/Caspase8+ and 7AAD+/Caspase+ events presented both in histogram and dot plot format.
(B) Summary bar charts comparing levels of activated caspase 8 in global versus CMV and
HBV-specific CD8+ T cells within the same patients with CHB (n=4). (C) Representative FACS plots and isotype control from a healthy individual and a CHB patient showing expression of
TRAIL-R2 on global peripheral CD8+ T cells and summary data form healthy n=18 and CHB n=27 patients (D) FACS plots depicting identification of virus-specific CD8+ T cells ex vivo via mutlimer staining from a representative CHB patient and gating strategy showing expression of TRAIL-R2 on virus specific cells. A control multimer was used to help identify the virus specific populations. (E) Paired data showing expression of TRAIL-R2 on HBV vs. CMV virus- specific CD8+ T cells from (n=9) CHB patients. (F) Representative FACS plot from an individual with resolved HBV infection, showing gating for HBV-specific CD8+ T cells and
TRAIL-R2 expression. (G) Summary bar charts of TRAIL-R2 expression on HBV-specific CD8+ T cells from 7 individuals with resolved HBV infection and 9 CHB individuals. Bars represent the mean±SEM.*P < 0.05, **P < 0.01.
151 4.5 Further upregulation of TRAIL-R2 on intrahepatic CD8+ T cells in CHB
Since the TRAIL-R2 death receptor was upregulated in CHB but largely confined to HBV- specific T cells, we hypothesised that its expression might be further enriched in the liver, the site of HBV replication. To determine whether TRAIL-R2 was expressed on CD8+ T cells infiltrating the liver, we initially examined paraffin-embedded sections of HBV-infected livers by immunofluorescence. Using this approach we noted clear co-staining of CD8+ T cells in the liver with TRAIL-R2 (Fig. 4.5A). We expanded on these data by extracting liver-infiltrating lymphocytes from surplus liver biopsy tissue, allowing flow cytometric comparison of TRAIL-
R2 expression on paired circulating and intrahepatic CD8+ T cell samples. In 19 out of 21 patients with CHB, the expression of TRAIL-R2 on CD8+ T cells was further increased in the liver compared to the peripheral compartment (Fig. 4.5B, C). There was considerable variability in TRAIL-R2 expression levels on intrahepatic CD8+ T cells (Fig. 4.5C), and this was found to correlate positively with HBV viral load (Fig. 4.5D). These data suggested that the liver environment and HBV may both be factors driving upregulation of this death receptor.
To address this, we obtained surplus liver tissue from 7 patients with HCV and 8 individuals without viral hepatitis. Liver-infiltrating CD8+ T cells from controls without viral hepatitis expressed more TRAIL-R2 than peripheral CD8+ from healthy donors, but much less than those from HBV-infected livers (Fig. 4.5E). CD8+ T cells extracted from HCV-infected livers showed a level of TRAIL-R2 expression intermediate between control and HBV-infected livers (Fig. 4.5E). Levels of TRAIL-R2 were lower on CD8+ T cells from HCV than HBV-infected livers despite the former group tending to be more inflamed (based on ALT levels, Tables 4.2 and 4.3). Levels of TRAIL-R2 were not simply a reflection of the degree of T cell activation; significantly more TRAIL-R2 was co-expressed by the HLA-DR+ (activated) fraction of CD8+ T cells extracted from HBV-infected livers compared to HCV or control livers (Fig. 4.5F).
Although we did not detect any significant differences in the expression of TRAIL-R1 in peripheral blood, the expression on intrahepatic lymphocytes was also significantly higher in
152 6 patients with available paired samples (Fig.4.5G). TRAIL-R3 was hardly detectable on the surface of peripheral T cells from healthy individuals and CHB patients and expression of
TRAIL-R4 was not significantly different between healthy controls and patients tested (data not shown). Due to limited intrahepatic samples a comprehensive analysis of all TRAIL receptors was not possible.
153
154
Figure 4.5. Intrahepatic CD8+ T cells in CHB patients have upregulated expression of TRAIL- R2
(A) Representative example of immunostaining of paraffin embedded liver tissue derived from a CHB patient undergoing diagnostic biopsy, showing CD8+ T cells and TRAIL-R2 co- localisation by immunofluorescence. Bar, 20μm. (B) Representative FACS plots from a CHB patient showing ex vivo expression of TRAIL-R2 on global peripheral and intrahepatic CD8+ T cells. (C) Ex vivo TRAIL-R2 expression on paired peripheral and intrahepatic global CD8+ T cells from (n=21) patients with CHB. (D) Correlation of TRAIL-R2 expression on intrahepatic
CD8+ T cells and HBV Viral Load. Spearman r= 0.4870, P value (two-tailed) = 0.02 (E)
Comparison of TRAIL-R2 expression on intrahepatic CD8+ T cells from 8 patients with non- viral hepatitis (control), 7 HCV infected patients and 21 CHB patients. (F) Co-staining for
TRAIL-R2 and HLADR gated on intrahepatic global CD8+ T cells directly ex vivo from a CHB patient, a HCV patient and a non-viral hepatitis control patient and summary data from
(n=5) control, (n=6) HCV and (n=10) CHB patients. (G) Representative FACS plots from a CHB patient showing ex vivo expression of TRAIL-R1 on global peripheral versus liver CD8+ T cells and isotype control and paired summary data form n=6 CHB patients. ***P < 0.001, **P <
0.01, *P < 0.05.
155 4.6 TRAIL-R2 expression: a hallmark of T cells encountering their cognate antigen in the HBV-infected liver
To further explore the characteristics of global intrahepatic CD8+ T cells bearing TRAIL-R2, we compared the expression of a panel of phenotypic markers on TRAIL-R2 high, low and negative fractions (Fig. 4.6A,B). TRAIL-R2 expressing CD8+ T cells were enriched for the activation marker CD38 (in line with the co-staining with HLA-DR demonstrated in Fig. 4.5F) and the apoptosis marker Annexin V, but not for the exhaustion marker PD-1 (Fig. 4.6A).
Cells expressing high levels of TRAIL-R2 were less likely to be found in the CD57+ and CD27-
CD45RA+ subsets and were instead mainly of a central memory phenotype (Fig. 4.6A,B).
These data suggest TRAIL-R2 expressing CD8+ T cells are activated, apoptotic responses rather than exhausted or senescent populations.
The large proportion of total intrahepatic CD8+ T cells bearing TRAIL-R2 in CHB led us to question whether its expression was restricted to HBV-specific T cells. In 5 HLA-A2+ individuals from whom sufficient liver-infiltrating lymphocytes could be obtained, responses directed against HBV or CMV were compared directly ex vivo by HLA-A2/peptide multimer staining. The expression of TRAIL-R2 was higher on intrahepatic CMV-specific CD8+ T cells than the low levels seen on CD8+ of this specificity in the periphery. However TRAIL-R2 was further enriched on intrahepatic HBV-specific CD8+ T cells (Fig. 4.6C). Upon recognition of their cognate antigen following overnight stimulation with HBV peptides, the majority of virus-specific CD8+ from HBV-infected livers expressed TRAIL-R2 (Fig. 4.6D). An upregulation of TRAIL-R2 was also observed following stimulation with CMV peptide on intrahepatic virus- specific CD8+ T cells from HBV infected patients (Fig. 4.6D summary bars). By contrast, CMV- specific CD8+ from control livers without hepatitis failed to express any TRAIL-R2 upon peptide stimulation (Fig. 4.D). Thus high TRAIL-R2 expression appeared to be a feature of T cells encountering their cognate antigen in the environment of an HBV-infected liver.
156
157
158 Figure 4.6. TRAIL-R2 expression is a feature of CD8+ T cells encountering antigen in the HBV-infected liver
(A) Representative gating strategy identifying intrahepatic TRAIL-R2+ high (red), TRAIL-R2+ low (black) and TRAIL-R2 negative (grey) CD8+ T cells from a CHB patient. Histograms and bars depict the proportions of CD38 (n=4), Annexin V (n=3, PD1 (n=6) and CD57 (n=8) expressed by each subset directly ex vivo. (B) The maturation status of intrahepatic TRAIL-
R2+ high (red), TRAIL-R2+ low (black) and TRAIL-R2 negative (grey) CD8+ T cells was analysed by co-staining for CD27 and CD45RA. Representative FACS plot and summary bars of frequencies of expression of TRAIL-R2+ high (red), TRAIL-R2+ low (black) and TRAIL-R2 negative (grey) CD8+ T cells with naïve, central memory (CM), effector memory (EM) and revertant (EMRA) phenotypes from n=4 CHB patients. (C) Summary bar charts comparing expression of TRAIL-R2 on global intrahepatic CD8+ T cells, CMV-specific and HBV- specific
CD8+ T cells. Virus-specific CD8+ T cells were identified directly ex vivo by multimer staining in (n=5) HLA-A2+ CHB patients with available liver biopsies. (D) Representative examples of the gating strategy from a control patient with no evidence of viral hepatitis (control liver) and a CHB patient (HBV liver) showing expression of TRAIL-R2 on intrahepatic global CD8+ T cells and virus-specific CD8+ T cells identified via IFN-γ staining following overnight stimulation with CMV peptide and HBV overlapping peptides (OLP) respectively. Summary bar charts comparing expression of TRAIL-R2 on CMV-specific CD8+ T cells from control livers (n=5) vs. CMV and HBV-specific CD8+ T cells from (n=5) CHB patients. Bars represent the mean±SEM.*P < 0.05.
159 4.7 TRAIL blocking partially restores HBV-specific CD8+ T cells
We next sought functional evidence for the involvement of the TRAIL pathway in NK cell mediated deletion of virus-specific T cells. To do this we examined the impact of TRAIL blockade at the time of peptide stimulation of PBMC on HBV-specific CD8+ T cell responses in vitro. TRAIL blocking experiments were performed with a TRAIL-R2 Fc, compared with a control IgG1 Fc to exclude a non-specific effect (Fig. 4.7A). Depicted in Fig. 4.7A are representative FACS plots from a CHB patient and summary data from 9 CHB patients during short-term culture, showing enhanced responses upon TRAIL blockade. However, the effect of TRAIL blockade was less striking than that of NK cell depletion in the same patients (Fig.
4.7A), suggesting that NK cells were utilising additional pathways for deleting T cells.
Although T cells can express TRAIL in some circumstances (Mirandola, Ponti et al. 2004;
Janssen, Droin et al. 2005), in our cohort TRAIL expression was confined to NK cells (Fig.
4.7B,C) and we could therefore assume that TRAIL blockade of PBMC was acting on NK cells.
In support of this approach, TRAIL blocking of PBMC depleted of NK cells did not show any further increase in the responses rescued above that observed upon NK depletion alone (Fig.
4.7A).
No significant recovery of CMV-specific CD8+ T cells from the same patients with CHB was observed upon TRAIL blockade (Fig. 4.7D), in keeping with the low levels of TRAIL-R2 expressed on these cells in the circulation and the lack of their reconstitution upon NK cell depletion. Further support for the preferential deletion of a selective subset of TRAIL-R2- bearing T cells by NK cells came from the finding that the percentage of global CD8+ T cells expressing TRAIL-R2 was increased following depletion of NK cells from PBMC cultures (Fig.
4.7 E).
160
161
Figure 4.7. Partial recovery HBV-specific CD8+ T cells to TRAIL blockade
(A) Representative FACS plots from a CHB patient following short-term peptide stimulation of PBMC, in the absence of NK cells and in the presence of TRAIL-R2/Fc blocking or IgG1-Fc control. Plotted are summary paired data from (n=9) CHB patients. (B) Summary data of ex vivo TRAIL expression on peripheral NK cells and CD3+ T cells from the cohort of patients with CHB included in this study. (C) Representative example of TRAIL expression on NK cells following ex vivo staining of PBMC, and on NK cells enriched by magnetic beads separation from the same CHB patient. (D) Representative FACS plot from a CHB patient and summary data from (n=6) patients showing the effect of TRAIL-R2 Fc addition at the time of PMBC stimulation with CMV peptide during short-term culture. (E) Representative example from a
CHB patient demonstrating the expression of TRAIL-R2 on global CD8+ T cells in the presence or absence (ΔNK) of NK cells following short term culture with HBV peptides and summary data from (n=19) CHB patients. Bars represent the mean ±SEM. ***P < 0.001, **P < 0.01, *P
< 0.05.
162 4.8 Overnight rescue of intrahepatic HBV-specific T cells by TRAIL blockade
NK cells constitute up to 40% of the lymphocytic infiltrate within the liver, the site of HBV replication. We have previously shown that intrahepatic NK cells in patients with HBV- related inflammation are highly activated and express increased levels of TRAIL compared to circulating NK cells, whereas intrahepatic CD3+T cells express little TRAIL (Dunn, Brunetto et al. 2007) (Fig. 4.8A). The extensive sinusoidal network in the liver forms a unique vascular bed with a narrow lumen and sluggish blood flow. We postulated that this would facilitate close associations between NK cells and T cells as they infiltrated the liver. To investigate this, we performed staining of paraffin-embedded liver sections from patients with CHB by immunohistochemistry. Using this approach we were able to visualise intimate contact between NK cells and T cells in the HBV-infected liver sinusoids, as exemplified in Fig. 4.8B.
This supported the concept that the intrahepatic predominance of TRAIL expressing NK cells, in conjunction with the unique architecture of the liver encouraging close contact between lymphocyte subsets, would promote the deletion of TRAIL-R2-bearing T cells.
To test whether the T cells infiltrating HBV-infected livers were subject to death receptor- mediated apoptosis, we stained them for caspase 8, which is activated by these pathways from the TNF superfamily (Sprick, Weigand et al. 2000). Caspase 8 was detectable in a proportion of CD8+ T cells isolated from HBV-infected liver biopsies; its expression was enriched in the expanded population of intrahepatic CD8+ T cells expressing TRAIL-R2 on their surface (Fig. 4.8C), in line with their enhanced expression of Annexin V (Fig. 4.6A).
These findings support the capacity of TRAIL-R2 to deliver an apoptotic signal to the large proportion of intrahepatic CD8+ T cells on which it is expressed in CHB.
To assess whether TRAIL blocking was capable of restoring virus-specific T cell responses in the liver, we examined paired samples from 9 HLA-A2- CHB patients from whom sufficient
163 liver biopsy tissue was available for functional experiments. Overlapping peptides (15mers) spanning the core protein of HBV were used for overnight stimulation and both peripheral and intrahepatic virus-specific T cell responses were assessed for IFN-γ production in the presence or absence of TRAIL blocking. Despite the fact that many of these cells were already poised to die, with activation of intracellular caspases, intrahepatic HBV-specific
CD8+ T cell responses could be augmented after just overnight TRAIL blockade in four out of nine patients (Fig. 4.8D). By contrast, the expansion of detectable HBV-specific T cell responses from PBMC could only be achieved after 10 days rather than overnight TRAIL blockade (data not shown). These results underscore the susceptibility of the enriched population of HBV-specific T cells in the liver compartment to NK cell TRAIL-mediated deletion.
164
165 Figure 4.8. Overnight recovery of intrahepatic HBV-specific T cells by TRAIL blockade
(A) Representative example of TRAIL staining on NK and CD3+ T cells in the periphery and liver from a CHB patient. (B) Examples of NK cells (NKp46 in blue) in intimate contact with
CD3+ T cells (red) in the sinusoidal spaces of a representative HBV-infected liver.
Immunohistochemistry was performed (by Dr G Reynolds, D. Adams group, Birmingham
University) on paraffin embedded HBV tissue. Bar, 20μm. (C) Representative FACS plot from a CHB patient and gating strategy showing expression of caspase 8 in intrahepatic TRAIL-R2+
CD8+ T cells ex vivo and summary bar charts comparing expression of caspase 8 in global
CD8+ T cells vs. TRAIL-R2+ CD8+ T cells (n=5). Only live events were analysed. (D)
Representative FACS plots showing response to TRAIL blockade following overnight incubation of intrahepatic cells with overlapping (OLP) HBV peptides and individual intrahepatic responses from 9 CHB patients with available liver tissue.
166 Discussion
Chronic infection with HBV is characterised by a number of T cell intrinsic and extrinsic defects, culminating in a profound depletion of the HBV-specific CD8+ T cell responses that constitute a crucial component of antiviral defence (Boni, Fisicaro et al. 2007; Lopes, Kellam et al. 2008; Protzer, Maini et al. 2012). In this study we focused on the role of NK cells in
HBV-specific T cell modulation. Our results demonstrate that NK cells can limit virus-specific
T cell responses, contributing to the apoptotic predilection of these cells. NK cells from patients with CHB can mediate these effects without the need for prior activation and at physiological ratios. NK-cell mediated regulation of T cell responses was found to be mediated in part through TRAIL dependent cytotoxicity. TRAIL is predominately expressed on NK cells in HBV infection and has been shown to amplify hepatocyte damage during disease fluctuations (Dunn, Brunetto et al. 2007). Here we show that HBV-specific CD8+ T cells, particularly those infiltrating the liver, upregulate the TRAIL-R2 death-inducing receptor. Therefore, NK cell mediated deletion of virus-specific T cells via TRAIL may perpetuate viral persistence and immune mediated pathology, especially within the inflammatory environment of the intrahepatic compartment.
Mounting evidence supports that NK cells have an important immunoregulatory role
(Martin-Fontecha, Thomsen et al. 2004) (Morandi, Bougras et al. 2006) (Zingoni, Sornasse et al. 2005), in addition to a direct antiviral function in the context of the early response to acute infection (Vivier, Tomasello et al. 2008). Our data are the first to identify a pathway for direct NK cell regulation of T cells in a human persistent viral infection; the presence of NK cells was found to specifically limit HBV-specific CD8+ T cell responses. These results complement existing evidence underscoring a detrimental role of NK cells on the developing antiviral response in the mouse LCMV infection. In this model activated NK cells targeted activated T cells in a cytolytic manner through a direct target cell recognition mechanism or
167 in the absence of 2B4 restraining NK cells. As a consequence there was a significant loss of
LCMV-specific CD8+ T effector cells, leading to impaired virus elimination and altered immunopathogy (Lang, Lang et al. 2012). In a follow-up study Waggoner et al build a case for
NK cell mediated cytotoxicity against CD4+ T cells, an important population supporting the function of virus-specific CD8+ T cells that control LCMV pathogenesis and persistence
(Waggoner, Cornberg et al. 2012). Thus, depending on the context of infection and viral kinetics, NK cells may dictate viral clearance or immunopathology and bear direct relevance in human chronic virus infections with HBV but also HCV or HIV.
The contrasting effects observed with responses to unrelated viruses versus HBV-specific responses upon NK cell depletion within our cohort of patients, point towards differential receptor-ligand regulation and NK cell-T cell interaction depending on antigen specificity. NK cells can potentially have either a beneficial or deleterious role that may support or override the protective effect of MHC in maintaining self-tolerance according to the type of infection.
This concept is supported by population genetic studies showing that KIR haplotypes are under balancing selection and can be linked with resistance to infection (Khakoo and
Carrington 2006) (Alter, Martin et al. 2007) but also immunopathology and autoimmunity
(Kulkarni, Martin et al. 2008) (Parham 2005). Although speculative, it is possible that reported associations between KIR haplotypes and outcome of infections with HIV, HCV and
HTLV-1 (Khakoo, Thio et al. 2004; Alter, Heckerman et al. 2011; Seich Al Basatena,
Macnamara et al. 2011) may in part be determined by differential NK cell modulation of antiviral T cell responses. Interestingly, expression of inhibitory KIRs and their cognate HLA ligands has been associated with HCV clearance (Khakoo, Thio et al. 2004), delayed HIV progression to AIDS (Martin, Qi et al. 2007), and resistance to acquiring infection in sex workers chronically exposed to HIV (Jennes, Verheyden et al. 2006). However, in CHB the protective effect of MHC class I molecule expression may be inadequate for self tolerance in
168 the face of selective upregulation of receptors/ligands superseding control of self-killing. The role of KIRs has not been explored in HBV infection in this context.
In this study we have concentrated on the role of TRAIL, a key effector molecule expressed on NK cells, in regulating the antiviral response during CHB. The contribution of the TRAIL pathway in the immune response to viral infections and pathogenesis has been the focus of a number of studies (Cummins and Badley 2009). The ultimate antiviral or pro-viral effect of
TRAIL is likely determined by the specific virus and the overall cytokine environment of the host. Notably CD8+ T cell memory responses have been found to be regulated via TRAIL- mediated activation induced death in the absence of CD4+ T cell help (Janssen, Droin et al.
2005). Lack of CD4+ T cell help may be particularly relevant in the liver, where CTL numbers outweigh CD4+ T cells and antigen presentation is defective (Protzer, Maini et al. 2012). T cell fratricidal killing via TRAIL may, therefore, constitute an important pathway of down- modulating the antiviral immune response in CHB. In HBV infected individuals, TRAIL is predominately expressed on activated NK cells in both the peripheral and liver compartments (Dunn, Brunetto et al. 2007), supporting that NK cells may be the primary mediators of T cell death via this pathway. Moreover, in the current study, NK cells were found to be in intimate contact with T cells within the liver sinusoids in the HBV infected liver suggesting that these interactions may be intensified in the intrahepatic compartment.
Compared to soluble TRAIL, membrane bound NK TRAIL has greater pro-apoptotic potential and its expression is further upregulated in individuals with evidence of liver inflammation.
Although TRAIL+NK cells can protect against tumours in the intrahepatic environment
(Takeda, Hayakawa et al. 2001) and may have an important anti-fibrotic role in chronic HCV
(Glassner, Eisenhardt et al. 2012) more recent human studies have highlighted a pathogenic role for this pathway, which is not exclusively restricted to transformed cells (Sato, Niessner et al. 2006) (Herbeuval, Grivel et al. 2005). Thus in the inflamed HBV liver the prominence of activated NK cells bearing high levels of TRAIL may have the dual effect of both mediating
169 hepatocyte injury (Dunn, Brunetto et al. 2007) and of promoting death of T cells with upregulated TRAIL receptors.
Activated human T cells can express TRAIL receptors and although they are normally protected against the apoptotic effects of soluble TRAIL (Mirandola, Ponti et al. 2004), several studies suggest that viral infections can render lymphoid cells susceptible to TRAIL mediated cytotoxicity (Katsikis, Garcia-Ojeda et al. 1997) (Jeremias, Herr et al. 1998) (Miura,
Misawa et al. 2001). We found an increase in the expression of TRAIL-R2, which transduces apoptotic signals upon TRAIL binding, on peripheral T cells from CHB patients. TRAIL-R2 was found to be substantially higher on the surface of HBV-specific cells compared to CMV- specific cells, suggesting that the features of HBV-specific CD8+ T cells that target them for
NK cell mediated deletion are not shared by T cells of other virus specificities. TRAIL-R2 was further upregulated on activated apoptosis-prone intrahepatic T cells along with the expression of TRAIL-R1, which also has an intracellular domain and apoptosis-inducing capability. The expression of TRAIL-R2 was more pronounced in CHB than HCV-infected or other control liver samples. Ongoing high-level expression of TRAIL-R2-bearing T cells in the
HBV-infected liver is consistent with the continuous renewal of antigen-primed T cells in persistent viral infections (Vezys, Masopust et al. 2006) and with our phenotypic characterisation of these populations. Elevated levels of caspase 8, that lies immediately downstream of death receptor signalling, partly explain the incomplete recovery of virus- specific responses in the liver, suggesting that blockade at the receptor level is not sufficient to rescue antiviral CD8+ T cells from their apoptotic destiny. We would, however, anticipate that receptor blockade would be more a more effective approach in vivo, allowing rescue of newly generated antiviral CD8+ T cells from an early demise. We have previously demonstrated that pre-treatment of PBMC from HBV patients with a pancaspase inhibitor, prior to peptide stimulation, can rescue HBV-specific CD8+ T cells that have upregulated Bim
170 (Lopes, Kellam et al. 2008). TRAIL signalling can also lead to increased levels of Bim and it is, therefore, plausible that the two pathways converge, enhancing the apoptotic propensity of virus-specific cells. Susceptibility to apoptosis has been investigated in the context of other hepatotropic virus infections, such as HCV, highlighting the role of caspase 9 mediated T cell death and the importance of cytokine deprivation (Radziewicz, Ibegbu et al. 2008). Of interest, caspase 8 mediated death signalling was not detected in HCV infection in the same study, which tallies with our findings of lower TRAIL receptor expression on intrahepatic T cells from HCV infected patients.
A question remaining is what increases the expression of death inducing receptors on T cells in CHB? Our data demonstrated a positive correlation of TRAIL-R2 expression on intrahepatic
T cells with HBV viral load. Whilst this may reflect part of a homeostastic control mechanism to restrain activated T cells during acute HBV infection, it could serve as a double edge sword in chronic infection potentiating viral persistence. Disease flares with increasing viral load and ensuing liver inflammation may therefore lead to an accelerated consumption of T cells expressing TRAIL receptors targeted by NK cells. It is conceivable that antigen presentation in the tolerogenic liver environment is able to impose a TRAIL-R2-expressing phenotype on T cells, as described for the pro-apoptotic molecule Bim (Holz, Benseler et al.
2008). A further potential mechanism of modulation of TRAIL receptors could be the virus- encoded antigens, in line with evidence that HBV-encoded X antigen upregulates TRAIL-R2 on hepatocytes in vitro, predisposing to TRAIL induced apoptosis (Janssen, Higuchi et al.
2003). Future studies utilising our large cohort of patients in different clinical phases of HBV will help dissect the relative contributions of viraemia, antigenaemia and liver inflammation on the induction of death receptors on T cells. We have previously demonstrated that cytokines induced during HBV flares may act jointly to modulate expression of both death- inducing and decoy TRAIL receptors, thereby maximising hepatocyte damage. In particular,
171 IL-8 was found to increase expression of TRAIL-R2 on the surface of hepatocytes, whereas
IFN-α substantially decreased expression of the regulatory TRAIL-R4 that lacks an intracellular death domain (Dunn, Brunetto et al. 2007). Additionally, Reactive Oxygen
Species (ROS) have also been shown to regulate T cell apoptosis (Hildeman 2004) and influence the expression of TRAIL receptors (Kwon, Choi et al. 2008) and may represent an important mechanism in the liver.
It remains unclear whether expression of TRAIL-R4 and/or TRAIL-R3 is a key factor in fine tuning susceptibility to TRAIL. However, it has been suggested that the ratio of expression may be tilted towards transduction of apoptotic signals in situations of liver inflammation such as bile acid retention (Higuchi, Bronk et al. 2001) and viral hepatitis. Another possible mechanism determining the sensitivity or resistance to TRAIL cytotoxicity is the level of expression of c-FLIP (cellular Fas associated death domain protein FADD like IL-1 converting enzyme [FLICE]- inhibitory protein). High levels of c-FLIP inhibit apical caspase-8, rendering cells resistant to TRAIL-mediated apoptosis (Mirandola, Ponti et al. 2004). It is possible that dysregulated HBV-specific T cells, in addition to altered TRAIL receptor expression, may also exhibit a down modulation in the homeostatic levels of intracellular c-FLIP.
Although, we have delineated a role for the TRAIL pathway, our data suggest that additional receptor/ligand interactions could contribute to driving NK cell lysis of T cells in CHB. The capacity of NK cells to lyse LCMV-specific CD8+ T cells has been shown to be regulated by
2B4 (Waggoner, Taniguchi et al. 2010) or mediated via the activating receptor, NKG2D recognising ligands expressed on antigen-specific CD8+ T cells (Lang, Lang et al. 2012). Our findings in CHB of NK cell inhibition of T cell survival by a contact dependent mechanism that involves pancaspase activation underline the potential contribution of other death receptors such as the FasL/Fas and/or pathways triggering cytotoxicity. Thus, the influence of HBV
172 infection on other relevant pathways regulating NK cell cytotoxicity or apoptosis induction merits further investigation. The involvement of 2B4, a member of the signaling lymphocyte activation molecule (SLAM) family of CD2-related receptors, as an important determinant of effective regulation of chronic viral infections is gathering momentum and has received some attention in persistent infection with HBV. In a recent study (Sun, Fu et al. 2012) elevated levels of TGF-β during the immunotolerant phase of CHB were associated with low levels of expression of 2B4/SAP on NK cells and impaired effector function. However, 2B4 is also expressed on exhausted virus-specific CD8+ T cells in a number of infections including
HBV (Wherry, Ha et al. 2007; Waggoner and Kumar 2012) (Aldy, Horton et al. 2011)
(Bengsch, Seigel et al. 2010) (Raziorrouh, Schraut et al. 2010). Blockade of 2B4 interacting with CD48 has been shown to restore exhausted virus-specific CD8+ T cells in vitro in HBV infection (Raziorrouh et al 2010) or to have a variable effect on HCV-specific CD8+ T cell functionality depending on the levels of 2B4 and SAP expressed by T cells (Schlaphoff,
Lunemann et al. 2011). Thus, when interpreting these data one must consider carefully the potential differential effects of 2B4 expression by NK cells or CD8+ T cells on their specific effector functions and overall antiviral capacity. Although the nature of the dual function of
2B4 is still a contentious issue, improved understanding of how 2B4 influences both individual antiviral activities and NK cell -T cell communication will help clarify its role in CHB infection.
The recent studies in LCMV show that NK cells can regulate CD8+ T cell responses not only by direct killing (Waggoner, Taniguchi et al. 2010; Lang, Lang et al. 2012) as demonstrated in our experiments, but also via elimination of CD4+ T cells (Waggoner, Cornberg et al. 2012).
This finding may also be pertinent to the liver, where CD4+ T cell numbers are already low and priming defective (Wuensch, Spahn et al. 2010). It would be important to address whether NK cells can equally restrict CD4+ T cell responses in CHB. More recently APCs from
NK cell depleted mice were reported to stimulate T proliferation, in a fashion independent of
173 the effects of NK cells on CD4+ T cells during LCMV infection (Cook and Whitmire 2013).
Thus it was proposed that NK cells may be hampering APC function during persistent viral infections, promoting T cell exhaustion (Cook and Whitmire 2013). Whether this is an additional mechanism contributing to T cell dysfunction in CHB remains currently unknown.
Under conditions of continuous stimulation NK cell cells may also exert negative regulatory functions as demonstrated by their ability to produce IL-10 to control adaptive responses during viral and parasitic infections (Biron 2010) (Lee, Kim et al. 2009) (Perona-Wright,
Mohrs et al. 2009) (Vivier and Ugolini 2009) (De Maria, Fogli et al. 2007). The concept of regulatory NK cells (NKregs) is emerging in the literature but it remains unclear if only a distinct subset of NK cells has immunoregulatory properties. Deniz and co-workers addressed the effect of IL-10 producing NK cells on T cell proliferation. Both allergen and antigen induced proliferation was found to be suppressed by IL10+ NK cells (Deniz, Erten et al. 2008). Moreover, in a well-defined model of visceral leishmaniasis NKp46+CD3- NK cells were recruited in the spleen and into hepatic granulomas where they inhibited host protective immunity in an IL-10 dependent manner (Maroof, Beattie et al. 2008). It has been further suggested that the strength of the microbial challenge may be important in determining the overall outcome of the NK cell response in terms of which cytokine will be predominately produced (Perona-Wright, Mohrs et al. 2009). Given the elevated levels of IL-
10 in CHB infection, investigation of the immunoregulatory function of NK cells in terms of
IL-10 production in persistent HBV infection would be relevant.
In summary, we provide evidence of a new pathway, whereby activated NK cells expressing death ligands may excessively down-modulate antiviral immunity in CHB. Although, NK cell mediated deletion of activated T cells may represent a homeostatic control mechanism to prevent exuberant immune responses, it may be detrimental in chronic viral infections,
174 shifting the balance in the on-going battle between the virus and adaptive immune responses. Our findings may also explain the divergent effects of exogenous IFN-α used in the treatment of CHB. IFN-α inhibits virus replication but also potently activates NK cells and upregulates TRAIL on their cell surface (Micco, Peppa et al. 2012). It would be of interest to investigate whether the observed limited T cell expansion observed during IFN-α treatment is NK cell mediated. NK cell activity can therefore be disadvantageous in persistent infection with HBV, regulating both the effector immune response and degree of immunopathology by utilising the same death ligands. With normal NK cell antiviral potential circumvented in chronic infection and a bias towards a pathogenic role, our data have implications for the development of future immunotherapeutic strategies. Attempts to block TRAIL therapeutically should be tempered with concerns over the potential beneficial role of this pathway against tumours and liver fibrosis (Takeda, Hayakawa et al. 2001) (Glassner,
Eisenhardt et al. 2012). With these considerations in mind, the timing of TRAIL blockade in the liver would need to be carefully considered. A TRAIL blocking strategy as a short-term adjuvant treatment to the immunotherapy of HBV may have the dual effect of promoting beneficial virus specific responses whilst minimising liver inflammation.
175 5. Ongoing work & Future Directions
Summary of work presented and unanswered questions
The work presented in this thesis contributes to the understanding of the antiviral and pathogenic role of NK cells in chronic infection with HBV and of the potential mechanisms that perpetuate infection.
In chapter 3, we explored the effector function and antiviral potential of NK cells in a large cohort of chronically infected HBV patients. Despite the observed expansion of the CD56bright subset, upregulation of TRAIL and conserved cytotoxicity, there was a selective defect in the capacity of NK cells to produce IFN-γ, one of the key cytokines implicated in non-cytolytic
HBV clearance. We were able to demonstrate that the immunosuppressive cytokine IL-10, which was found to be elevated in CHB patients with active disease was able to reproduce this functional defect in vitro. Importantly, TRAIL expression, along with cytolytic ability were maintained in the presence of IL-10. Although potent oral antiviral therapy was able to restore NK cell subsets and phenotype, it failed to normalise IL-10 levels or the capacity of
NK cells to produce IFN-γ. Blockade of IL-10 +/- TGF-β restored the ability of peripheral and intrahepatic NK cells of patients with CHB to produce IFN-γ, thereby enhancing their non- cytolytic antiviral capacity. This functional divergence of NK cell effector function is in agreement with further published evidence, supporting an NK cell bias towards heightened cytolytic function in CHB and necroinflammation.
Peg-IFN-α, which constitutes one of the most successful treatments in chronic HBV infection, has potent immunoregulatory effects and has been shown to enhance NK cell effector function in murine models and upregulate TRAIL expression in CHB patients (Dunn,
Brunetto et al. 2007) (Nguyen, Salazar-Mather et al. 2002). Whether therapeutic IFN-α is able to drive the proliferation of NK cells and restore their capacity to produce IFN-γ, was
176 addressed by Micco et al in our group. This recent study demonstrated that IFN-α can collectively increase proliferation (by inducing IL-15), activation status and antiviral function of the NK CD56bright subset, in contrast to its depleting effects on CD8+ T cells (Micco, Peppa et al. 2012). NK cell TRAIL expression and IFN-γ production peaked at the last treatment time point, which related temporally with viral nadir within individuals (Micco, Peppa et al. 2012).
Although no reduction in circulating IL- 10 levels were observed during therapy, the ability of
IFN-α to boost NK cell function could override the immunosuppressive effects mediated by
IL-10. A larger study will help delineate whether the expansion of functional NK cells are predictive of clinical response, and refine our ability to identify an NK cell subset with prognostic relevance to treatment outcome. Along the same lines it would be important to gain further knowledge on the different effects imposed by IFN-α in NK cells at a transcriptional level.
Although the cytokine environment fine-tunes the action of NK cells, it is likely that a number of mechanisms including receptor modulation and interaction with DCs contribute to the overall dysregulation of NK cells in CHB (Woltman, Op den Brouw et al. 2011). The potential crosstalk between Kupffer cells and NK cells is an area that deserves further attention. Interestingly upregulation of the co-inhibitory molecule Tim-3 on NK cells has been reported to mediate suppression of their function during CHB (Ju, Hou et al. 2010).
Work from our group has identified strong expression of its ligand, galectin-9, on Kupffer cells (Nebbia, Peppa et al. 2012). In addition to the ability of Kupffer cells to influence NK cells through production of immunosuppressive cytokines such as IL-10 and TGF-β that can tolerise local NK cells (Tu, Bozorgzadeh et al. 2008), bidirectional communication through receptor-ligand interactions could mediate additional effects. Future work is required to address these potential interactions.
177 In chapter 4, we investigated the immunoregulatory role of NK cells in terms of restraining adaptive antiviral immunity. Depletion of NK cells was shown to restore virus-specific CD8+ T cell responses; an effect limited to HBV rather than unrelated viruses within the same chronically infected patients. This effect was found to be partially mediated via the TRAIL pathway. Overall these findings highlight a potentially novel mechanism silencing virus- specific T cells in chronic HBV infection. Our data provide new insights into the interactions between NK cells and T cells, which may be most pertinent in the liver. Figure 5.1 is a schematic representation summarising the findings presented in this thesis.
HBV%infected%
HBV hepatocytes% HBV! HBV! !
HBV# HBV# HBV# HBV#
Viral%% Liver%injury% control% IL;10% TGF;β"
Impaired% Viral%control% NK# CD8# NK# CD8# ?%
?% Help% DC# CD4# CD4#
Figure 5.1 Working hypothesis of the antiviral and regulatory role of NK cells in CHB. Question marks denote unknown interactions between NK cells and CD4 T cells and dendritic cells (DC).
178 The work outlined in chapter 4, generated a number of important and interesting questions.
Specifically:
• Which features of activated T cells trigger susceptibility to NK cell lysis? Is this
phenomenon restricted to CD8+ or does it extend to CD4+ T cells?
• What receptor ligand interactions control the specificity of NK cell lysis of T cells?
• Which mechanisms police the immunoregulatory functions of NK cells?
• What cytokines/factors stimulate NK cell killing of T cells?
• Is there a role for other mechanisms of NK cell regulation such as production of IL-10
and crosstalk with other immunoregulatory populations?
• What is the effect of viral kinetics and role of NK cells in the generation/regulation of
T cell memory (ie acute infection or during vaccination)
Although this is not an exhaustive list, efforts to address these points will ultimately lead to greater insights into the biological role of NK cells and increase the current knowledge of host-virus relationships. We will present below preliminary data tackling some of the questions raised, along with a general outlook into other human chronic viral infections.
Are CD4+ T cells amenable to NK mediated deletion in CHB infection and is the TRAIL pathway involved?
The recent studies in the mouse LCMV infection concluded that NK cell cytotoxicity is required to mediate its negative effects on antiviral T cell immunity, however the immune cell target was different. Lang et al showed through the use of different LCMV strains and NK cell depleted or deficient mice that NK cell perforin-dependent lysis of CD8+ T cells led to viral persistence and immunopathology (Lang, Lang et al. 2012). Waggoner et al demonstrated that during infection with LCMV clone 13, NK cells selectively targeted CD4+ T cells through cytotoxicity, regulating the CD4+ T cell mediated support for virus–specific
179 CD8+ T cells (Waggoner, Cornberg et al. 2012). In the absence of 2B4, NK cells were also able to exhibit enhanced cytotoxicity against CD8+ T cells (Waggoner, Taniguchi et al. 2010). Of note the use of a modified in vivo cytotoxicity assay demonstrated that viral infections, in addition to potent pro-inflammatory stimuli, can generate rapid NK cell mediated killing of activated CD4+ T cells and to a lesser extent CD8+ T cells, suggesting differential susceptibility to NK cell mediated elimination (Waggoner, Cornberg et al. 2012).
When considering the potential targets of NK cell mediated killing one must take into consideration the complexities associated with different experimental systems, along with issues arising from specific timing of delivery of cytotoxicity and sensitivity of the particular target cell. Nonetheless elimination of CD4+ T cells by NK cells in CHB is an interesting proposition.
Our preliminary experiments with NK cells purified directly ex vivo from a high level HBV carrier induced pancaspase activation after 4 hours co-incubation in a small population of autologous CD4 and CD8+ T cells. This is consistent with the low circulating frequencies of
HBV-specific T cells that we postulate may be susceptible to this form of deletion. By contrast, NK cells isolated from a healthy donor had no effect on the survival of T cells (Fig
5.2)
180 * 3
2 MFI 1 FOLD CHANGE FOLD
0 HEALTHY CHB HEALTHY CHB CD8 CD4 n=4
Figure 5.2 Ex vivo killing capacity of NK cells
NK cells purified directly ex vivo were re-added to autologous T cells at a ratio of 1:1 (roughly equivalent to the intrahepatic ratio of NK to T cells). After 4 hours, the degree of caspase activation was determined by flow cytometry using the carboxyfluorescein-FLICA apoptosis detection kit.
Summary data of fold change increase in the FLICA MFI of CD8 and CD4+ T cells from 4 healthy and 4
CHB patients (fold change: FLICA MFI of T cells following re-addition of NK cells divided by FLICA MFI of T cells alone). Significance testing was done using the Mann-Whitney test (*p<0.05; **, p<0.001;
***p<0.001).
These observations suggested that CD4+ T cells may also be vulnerable to NK cell mediated deletion. Although in chapter 4 we concentrated our efforts on CD8+ T cells, we were also able to assess the impact of depletion of NK cells from CHB PBMC on frequencies of HBV- specific CD4+ T cells following stimulation with OLP in a small number of patients.
Elimination of NK cells from these PBMC cultures also increased the proportion of virus- specific CD4+ T cells identified by intracellular staining for IFN-γ following short-term culture in selected patients (Fig 5.3).
181 PBMC Δ NK
Figure 5.3 Increased proportions of activated HBV-specific CD4+ T cells in the absence of NK cells
Representative FACS plots showing HBV-specific CD4+ T cell identified by IFN-γ staining, following short-term culture with overlapping (OLP) HBV peptides in the presence and absence of NK cells
(upper panel) and levels of activation of CD4+ T cells by HLA-DR expression (lower panel) from a representative CHB patient.
Recently an in vitro study highlighted the role of TRAIL in the regulation of activated autologous CD4+ T cells. Activated CD4+ T cells were reported to upregulate both TRAIL-R1 and TRAIL-R2 increasing their susceptibility to NK TRAIL-mediated lysis (Nielsen, Odum et al.
2012). Thus it is tempting to speculate that the TRAIL pathway may also have an effect on
CD4+ T cell survival in the setting of chronic HBV infection.
Notably, we observed higher expression of TRAIL-R2 on CD4+ T cells in CHB compared to healthy individuals in peripheral blood, which was further increased in the liver
182 compartment, but to a lesser extent compared to CD8+ T cells. The expression of TRAIL-R2 on intrahepatic CD4+ T cells was also higher compared to global T cells from control liver biopsies and patients with chronic HCV and available liver tissue (Fig 5.4A,B,C). No correlation with HBV viral load was observed (Fig 5.4D)
A" B" *** 1.5 ** 35 30
1.0 25 20 15 0.5 10 %CD4TRAIL-R2 %CD4TRAIL-R2+ 5 0.0 0 HEALTHY CHB PBMC IHL n=18 n=21 n=21
C" * D" 15 * 11 R= 0.2818 10 9 8 10 7 6 5 5 4
LogHBVDNA 3
%CD4TRAIL-R2+ 2 1 0 0 CTR HCV CHB 0 10 20 30 40 n=6 n=7 n=21 %CD4TRAIL-R2
Figure 5.4 Increased TRAIL-R2 expression on CD4+ T cells in CHB patients
(A) Ex vivo TRAIL-R2 expression on peripheral CD4+ T cells from n=18 healthy controls and n=21 CHB patients. (B) paired peripheral and intrahepatic global CD4+ T cells from (n=21) patients with CHB. C)
Comparison of TRAIL-R2 expression on intrahepatic CD4+ T cells from 6 patients with non-viral hepatitis (control), 7 HCV infected patients and 21 CHB patients. (D) Correlation of TRAIL-R2 expression on intrahepatic CD4+ T cells and HBV Viral Load. Spearman r= 0.2818
Addition of TRAIL-R2 Fc blocking at the time of OLP stimulation of IHL led to increased proportions of virus-specific CD4+ T cells in 3 out of 9 patients (Fig 5.5A,B). However, intrahepatic CD4+ T cells were also found to have increased levels of caspase 8 identified
183 directly ex vivo, explaining in part the limited capacity of TRAIL blocking to restore their antiviral potential (Fig 5.5C,D).
A" B" 8 OLP 7 OLP TRAIL Blocking 6 + γ 5 4 3 %CD4IFN 2 1 0 PT1 PT2 PT3 PT4 PT5 PT6 PT7 PT8 PT9 C" D" 80 70 60 50 40 30
%Caspase 8 20 10 0 Global CD4 CD4TRAILR2+ n=3
Figure 5.5 Limited recovery of intrahepatic HBV-specific T cells by TRAIL blockade
(A) Representative FACS plots showing response to TRAIL blockade following overnight incubation of intrahepatic cells with overlapping (OLP) HBV peptides and (B) individual intrahepatic responses from
9 CHB patients with available liver tissue. (C) Representative FACS plot from a CHB patient and gating strategy showing expression of caspase 8 in intrahepatic TRAIL-R2+ CD4+ T cells ex vivo and summary bar charts comparing expression of caspase 8 in global CD4+ T cells vs. TRAIL-R2+ CD4+ T cells (n=3).
Only live events were analysed.
The effect of NK cells on antiviral CD4+ T cells is the focus of current studies in the group.
Additional experiments will help define further the action of NK cells on CD4+ T cells survival and residual effect on CD8+ T cells.
184 Additional pathways mediating deletion
Our data suggest that additional receptor/ligand interactions are involved in mediating NK cell deletion of T cells in CHB. Besides TRAIL, TNF itself and the CD95 (Fas/APO-1) ligand
(FasL/Apo1L), belong to the subfamily of ligands that is responsible for extrinsic induction of cell death. It would be of interest to determine the relative contribution of these pathways to NK cell-mediated killing of T cells. This can be achieved by examining the expression levels of these molecules on NK cells and their corresponding receptors on T cells from both peripheral blood and the liver of CHB patients. In the killing experiments already described the addition of blocking antibodies will help delineate a role for these pathways.
A number of mouse and human studies have previously indicated the potential involvement of the NKG2D pathway in restraining T cell responses and promoting T cell homeostasis.
NKG2D binds to a number of different inducible self-proteins including MICA/B and ULBP1-6 in humans, and H60 (a-c), Rae (a−e) and MULT1 in mice (Eagle and Trowsdale 2007).
Whereas expression of the ligands for NKG2D is low on healthy cells, it can be induced on infected, transformed or ‘stressed’ cells. Although resting T cells do not express NKG2D ligands (NKG2DLs), different stimuli have been shown to upregulate them on their cell surface. Additionally, chronic in vivo antigenic stimulation of CD4+ T lymphocytes has been reported to enhance levels of expression of H60 and MULT1 (Noval Rivas, Hazzan et al.
2010). T cell activation and upregulation of NKG2DLs increases autologous NK cell mediated lysis in both animal models and human in vitro studies (Rabinovich, Li et al. 2003) (Cerboni,
Zingoni et al. 2007). A recent study further demonstrated that NKG2D, LFA-1 and NKp46 are involved in NK cell mediated cytotoxicity against activated autologous CD4+ T cells; notably both NK cell subsets were equally found to mediate this effect (Nielsen, Odum et al. 2012).
NKG2D-dependent killing of activated CD8+ T cells has also been described to reduce the memory T cell compartment (Soderquest, Walzer et al. 2011).
185 In the setting of viral infections, it is now increasingly recognised that NKG2D plays an immunoregulatory role in addition to its crucial function in the direct recognition and lysis of infected cells (Lanier 2008) (Rossini, Cerboni et al. 2012) (Zingoni, Ardolino et al. 2012).
Previous studies established that the presence of NK cells can limit CD4+ and CD8+ T cell responses and proliferation during MCMV and LCMV infection (Su, Nguyen et al. 2001)
(Andrews, Estcourt et al. 2010) (Mitrovic, Arapovic et al. 2012). More recently, the capacity of NK cells to lyse LCMV-specific T cells has been shown to be dependent on NKG2D (Lang,
Lang et al. 2012) and to contribute to viral persistence and disease pathology, making this target cell recognition mechanism particularly attractive (Lang, Lang et al. 2012). The role of
NKG2D along with other activating receptors such as DNAM, in persistent infection with HBV remains to be determined. Our preliminary data on the NKG2D pathway, outlined below, suggest that it may be a relevant avenue for future investigation.
From our extensive CHB cohort, we have established that there is high expression of NKG2D on the surface of NK cells both in the periphery and in the intrahepatic compartment of CHB patients (Fig 5.6). Conversely very little is known about the expression of NKG2DLs in CHB.
186 Periphery 100
80
60
40
20 %NKG2D+ NK cells
0 Healthy Lo ALT Hi ALT Liver-CHB
150 100
75 100
50 MFI 50 25 %NKG2D+ NK cells
0 0 PBMC IHL PBMC IHL
Fig 5.6 Conserved expression of NKG2D on NK cells from CHB patients
Representative FACS plot comparing ex vivo expression of NKG2D on NK cells on a healthy control versus a CHB patient and summary data. Summary dot plots include categorisation of CHB patients according to liver inflammation (ALT) levels. Lo ALT refers to ALT levels <50; HI ALT refers to ALT levels
>50 (upper panel). The lower panel depicts a representative FACS plot comparing NKG2D expression on peripheral compared to liver NK cells from a CHB patient. Paired data are also shown for CHB patient (%andMFI).
Screening using reporter constructs transfected with a panel of 11 activatory NK receptors
(collaboration with Prof Trowsdale, Cambridge (Fig 5.7)) identified an increase in NKG2DLs on T cells in CHB versus controls (Fig 5.8). Higher expression was noted on CD4+ T cells (Fig
5.8). We therefore hypothesised that expression of NKG2DLs by T cells in CHB allows them to activate NK cells through NKG2D and induce granule exocytosis, thereby constituting an additional pathway for T cell deletion.
187 Subclone%DAP10%adaptor%in%pMX;neo%vector% NFAT%GFP(
Infect%2B4%cell%line%and%select%NeoR%clone%
NFAT%GFP( Subclone%NKG2D%into%pMX;puro%vector%and%infect%adaptor/2B4%cells%
FACS%sort%posi,ve%cells%and%screen%clones%with%GFP% NFAT%GFP( expression%by%an,body%cross;linking%
Fig 5.7 Depiction of 2B4 model system used. Kindly provided by Dr C Chang (Cambridge University)
188 Fig 5.8 NKG2DL expression on PBMCs from CHB patients and healthy controls based on GFP expression on 2B4 cell line.
When these reporter cells were co-cultured for 18 hours with PBMC or purified cells they induced GFP expression (measured by flow cytometry) indicating the presence of ligand/s. PBMC from patients with CHB were only able to bind to NKG2D receptor, inducing GFP expression. FACS plots show the expression of NKG2D ligands (%GFP expression) in PBMC from 2 patients with active CHB and one healthy individual (upper panel) and cumulative data from patients with CHB (High and low ALT) compared to healthy individuals (Upper panel dot plots). The expression of NKG2D ligands (%GFP expression) from purified CD4+ T cells (lower panel) from the same 2 patients with active CHB and a one healthy individual are depicted in the lower panel along with a negative control with reporter cells alone. High ALT group was defined if ALT>60.
To complement the above, a panel of directly conjugated antibodies for individual NKG2DLs will be used to probe which particular ligand is upregulated on the surface of global and virus specific T cells in HBV infection; these results will be stratified according to disease activity and clinical profile of the patients. Available intrahepatic samples will be used to compare expression in the liver and peripheral compartment, where feasible. Functional killing experiments in the presence of blocking antibodies for NKG2D or selective blocking of perforin will help address the role of this pathway in NK cell mediated cytotoxicity.
Utilising the same T cell hybridoma system carrying the GFP reporter construct transfected with GFP, we were also able to demonstrate an upregulation of NKG2DLs on purified CD14 cells from CHB patients. This opens up an additional area of investigating cognate interactions between NK cells and CD14 cells in CHB that may constitute a pathway of monocyte editing. An alternative possibility is that CD14 cells are not directly susceptible to direct NK cell killing but upregulate molecules able to activate NK cells to kill autologous T cells through three-way interactions.
189 Future Outlook: Explore the role of NK cells in antiviral immunity in other chronic viral infections
Our work, in addition to evidence stemming from animal models, highlights the regulatory potential of NK cells and their contribution to viral pathogenesis and persistence. These emerging data challenge the paradigm of NK cell function during viral infections and may be relevant in the context of other chronic infections such as HCV and HIV. It would therefore be of interest to explore how this work translates into HCV and HIV and whether NK cell mediated modulation of T cell responses is a generic principle underlying viral infections that orchestrates the balance between viral elimination and development of chronicity with pathological sequelae.
The role of TRAIL in antiviral defense and pathogenesis of individual viral infections is clearly an important one that requires better definition. Evidence from TRAIL and TRAIL receptor knock-out mice examining the biological role of TRAIL to experimental infectious and inflammatory conditions suggest that it may have a dual function as both an innate effector mechanism in response to intracellular pathogens but also as a negative regulator of the immune response (Diehl, Yue et al. 2004) (Zheng, Jiang et al. 2004) (Zheng, Wang et al. 2004)
(Cummins and Badley 2009). Moreover, our study implicated TRAIL expressing NK cells in the negative regulation of antiviral T cell responses, which may be pertinent in both HCV and
HIV infection.
Investigating the direct contribution of NK cells in the massive culling of T cells in HIV infection is an exciting but challenging area, given that HIV-1 infects CD4+ T cells. In untreated progressive HIV infection a large contributor to the inevitable decline of T cells is apoptosis (Cummins and Badley 2009). The involvement of the TRAIL pathway has been supported by a number of studies showing increased levels of soluble and membrane bound
190 TRAIL and TRAIL-R2 (DR5) on circulating lymphocytes in HIV infected individuals (Cummins and Badley 2009) (Funke, Durr et al. 2011) (Gougeon and Herbeuval 2012). Induction of
TRAIL through type I IFN on pDCs has been shown to transform them into killer cells (IKpDCs) capable of triggering apoptosis in both HIV infected CD4+ T cells but also uninfected bystander CD4+ T cells (Hardy, Graham et al. 2007) (Stary, Klein et al. 2009). This pathway may be more relevant in secondary lymphoid organs such as the tonsils where TRAIL+ lymphocytes (IKpDcs) were reported to be in close contact proximity to the apoptotic CD4+ T cells (Stary, Klein et al. 2009). Interestingly, a more recent study suggested a more elaborate interaction between pDC, primary T cells and NK cells, resulting in lysis of HIV-1 infected
CD4+ T cells through NK cell mediated cytotoxicity (Chehimi, Papasavvas et al. 2010). Thus the role of TRAIL is likely to be more complex than was originally proposed in HIV infection and the involvement of NK cells requires further careful dissection. Additional evidence implicates NK cells in the elimination of uninfected CD4+ T cells in HIV infection using alternative receptor/ligand interactions. This effect was found to be mediated by NKp44 expressed on NK cells recognising an HIV-1 induced cellular ligand for NKp44 (Nkp44L)
(Vieillard, Strominger et al. 2005). NK cells can therefore serve as a double-edge sword in
HIV infection, contributing to both disease clearance and immunopathogenesis.
As with HIV there are several lines of evidence supporting a role of TRAIL in HCV infection.
However the seemingly disparate findings reported on the involvement of TRAIL as a mediator of apoptosis and/or role in viral control make it difficult to evaluate the contribution of this pathway in HCV pathogenesis without further study (Saitou, Shiraki et al.
2005) (Mundt, Kuhnel et al. 2003) (Ahlenstiel, Titerence et al. 2010) (Varchetta, Mele et al.
2012). Thus, a potential involvement of NK TRAIL+ cells as negative immune regulators in
HCV remains to be identified.
191 Recent description of effects arbitrated through inhibitory/activating NK receptor–ligand interactions provide new awareness into potential pathways of regulation of NK cell responses. Certain KIR/HLA interactions have been associated with particular outcomes of
HIV and HCV infections as previously discussed. It would therefore be important to determine in future studies the effect of KIR diversity on NK cell mediated regulation of T cells. Of note, expression of inhibitory KIRs and their cognate HLA ligands has been associated with HCV viral clearance and delayed disease progression or resistance to infection with HIV (Khakoo, Thio et al. 2004) (Martin, Qi et al. 2007) (Jennes, Verheyden et al. 2006). Specifically, in HIV infection the interaction between the inhibitory KIR3DL1 and
HLA-Bw4 influences the rate of HIV progression. In HCV infection, homozygosity for the inhibitory NK cell KIR2DL3 receptor gene and its corresponding ligands (HLA-C group alleles) is associated with viral clearance and superior response to IFN-α therapy (Khakoo, Thio et al.
2004; Knapp, Warshow et al. 2010). Although the KIR2DL3:HLAC1 assortment is a relatively weak inhibitory interaction, it is tempting to speculate that inhibition or moderation of NK cell activity mediated by an inhibitory KIR could potentially improve virus-specific T cell responses. This is supported by a recent study from a cohort of HIV-1 infected elite controllers, where NK cell function and responsiveness correlated inversely with the degree of antiviral T cell responses (Tomescu, Duh et al. 2012). Our data derived from HCV infected patients studying the effect of NK cell depletion on the magnitude of virus-specific responses demonstrated a variable response on the numbers of HCV-specific T cells, which may reflect the presence or absence of various KIR receptors.
The precise mechanisms in place to regulate NK cell functions as well as the different pathways that may ‘condition’ them during different challenges are only beginning to emerge. Unraveling these could lead to potential new strategies of harnessing NK cell function, which may be beneficial in the context of persistent viral infections directing the
192 balance toward viral clearance.
193 List of publications and abstracts
1. Maini, MK, Peppa D. NK cells a double-edge sword in chronic hepatitis B virus infection. Front Immunol. 2013; 4:57. doi: 10.3389/fimmu.2013.00057. Epub 2013 Mar 1.
2. Dimitra Peppa, Upkar S. Gill, Gary Reynolds, Nicholas J.W. Easom, Anna Schurich, Lorenzo Micco, Gaia Nebbia, Harsimran D. Singh, David H. Adams, Patrick T.F. Kennedy, Mala K. Maini. Upregulation of a death receptor renders antiviral T cells susceptible to NK cell mediated deletion. J Exp Med. 2013 Jan 14;210(1):99-114. doi: 10.1084/jem.20121172. Epub 2012 Dec 17.
3. Nebbia G, Peppa D, Schurich A, Khanna P, Singh HD, et al. (2012) Upregulation of the Tim-3/Galectin-9 Pathway of T Cell Exhaustion in Chronic Hepatitis B Virus Infection. PLoS ONE 7(10): e47648. doi:10.1371/journal.pone.0047648
4. Dimitra Peppa, Mala K Maini. Pathogenesis of hepatitis B virus infection and potential for new therapies. British Journal of Hospital Medicine, Vol. 73, Iss. 10, 09 Oct 2012, pp 581 – 584
5. Micco L, Peppa D, Loggi E, Schurich A, Jefferson L, Cursaro C, Panno AM, Bernardi M, Brander C, Bihl F, Andreone P, Maini MK. Differential boosting of innate and adaptive antiviral responses during pegylated-interferon-alpha therapy of chronic hepatitis B. J Hepatol. 2012 Oct 6. pii: S0168-8278(12)00761-1. doi: 10.1016/j.jhep.2012.09.029. [Epub ahead of print]
6. Das A, Ellis G, Pallant C, Lopes AR, Khanna P, Peppa D, Chen A, Blair P, Dusheiko G, Gill U, Kennedy PT, Brunetto M, Lampertico P, Mauri C, Maini MK. IL-10-Producing Regulatory B Cells in the Pathogenesis of Chronic Hepatitis B Virus Infection. J Immunol. 2012 Oct 15;189(8):3925-35. doi: 10.4049/jimmunol.1103139. Epub 2012 Sep 12.
7. Nebbia G, Peppa D, Maini MK. Hepatitis B infection: current concepts and future challenges. QJM 105(2):109-113 Feb 2012.
8. Schurich A, Khanna P, Lopes AR, Han KJ, Peppa D, Micco L, Nebbia G, Kennedy PT, Geretti AM, Dusheiko G, et al. Role of the coinhibitory receptor cytotoxic T lymphocyte antigen-4 on apoptosis-Prone CD8 T cells in persistent hepatitis B virus infection. Hepatology 53(5):1494-1503 May 2011.
9. Peppa D, Micco L, Javaid A, Kennedy PT, Schurich A, Dunn C, Pallant C, Ellis G, Khanna P, Dusheiko G, et al. Blockade of immunosuppressive cytokines restores NK cell antiviral function in chronic hepatitis B virus infection. PLoS Pathog 6(12):e1001227 2010.
10. Dunn C, Peppa D, Khanna P, Nebbia G, Jones M, Brendish N, Lascar RM, Brown D, Gilson RJ, Tedder RJ, et al. Temporal analysis of early immune responses in patients with acute hepatitis B virus infection. Gastroenterology 137(4):1289- 1300 Oct 2009.
194
Conference Abstracts & Presentations to Learned Societies
1. D.Peppa, U.S. Gill, L., G Reynolds, N. J. W. Easom, Micco, A. Schurich, G. Nebbia, H. Singh, D. Adams, P.T.F. Kennedy, M.K. Maini. Regulation of adaptive responses by NK cells in chronic HBV infection. 2012 International Meeting The Molecular Biology of Hepatitis B Viruses, Oxford, UK. (oral)
2. D. Peppa, N. J. W. Easom, U.S. Gill, L. Micco, A. Schurich, G. Nebbia, H. Singh, W. Rosenberg, R. J. Gilson, P.T.F. Kennedy, M.K. Maini. Susceptibility of T cells to NK cell death ligand-mediated deletion in the liver of chronic hepatitis B virus (CHB) infected patients. 13th International Meeting of the society for Natural Immunity (NK 2012), Asilomar, California, USA. (oral)
3. N. J. W. Easom, D. Peppa, X. Tang, L. Pallett, C. Chang, J. Trowsdale, M.K. Maini. NK CELLS REGULATE T-CELL RESPONSES IN CHRONIC HEPATITIS B VIA NKG2D. 13th International Meeting of the society for Natural Immunity (NK 2012), Asilomar, California, USA.
4. Gill US, Papadaki M, Peppa D, Micco L, Li L, Ushiro-Lumb I, Foster GR, Maini MK, Kennedy PTF. MAXIMAL BOOSTING OF INNATE IMMUNITY DURING PEGYLATED INTERFERON-APLHA THERAPY IS REACHED AT 48 WEEKS IN E-ANTIGEN POSITIVE CHRONIC HEPATITIS B. JOURNAL OF HEPATOLOGY. 56: S175-S175. Apr 2012
5. Micco L, Peppa D, Loggi E, Schurich A, Cursaro C, Panno AM, Bihl F, Bernardi M, Andreone P, Maini MK. Pegylated interferon-alpha potently augments NK cell antiviral effector function in chronic hepatitis B. DIGEST LIVER DIS 43:S68-S69 Feb 2011
6. D. Peppa, U.S. Gill, L. Micco, A. Schurich, G. Nebbia, H. Singh, W. Rosenberg, R. J. Gilson, P.T.F. Kennedy, M.K. Maini. Susceptibility of T cells to death ligand-mediated deletion in the liver of chronic hepatitis B virus (CHB) infected patients. 2011 International Meeting The Molecular Biology of Hepatitis B Viruses, Orlando, USA (poster)
7. Schurich A, Khanna P, Lubowiecki M, Lopes AR, Peppa D, Micco L, Nebbia G, Singh H, Rosenberg W, Kennedy PTF, et al. ALTERING CO-INHIBITORY AND CO-STIMULATORY PATHWAYS TO RESTORE ANTI-VIRAL T CELL RESPONSES IN CHRONIC HBV INFECTION. JOURNAL OF HEPATOLOGY. 54: S125-S125. Mar 2011
8. Micco L, Peppa D, Loggi E, Schurich A, Carmela C, Panno AM, Bihl F, Bernardi M, Andreone P, Maini MK. PEGYLATED INTERFERON-ALPHA POTENTLY AUGMENTS NK CELL ANTIVIRAL EFFECTOR FUNCTION. JOURNAL OF HEPATOLOGY. 54: S122-S122. Mar 2011
9. Gill US, Micco L, Li L, Peppa D, Ushiro-Lumb I, Foster GR, Maini MK, Kennedy PTF. PEGYLATED INTERFERON ALPHA MODULATES INNATE IMMUNITY IN EAG POSITIVE CHRONIC HEPATITIS B AND DETERMINES CHANGES IN HBSAG QUANTIFICATION. JOURNAL OF HEPATOLOGY. 54: S32-S32. Mar 2011
10. Peppa D, Micco L, Schurich A, Nebbia G, Khanna P, Singh H, Rosenberg W, Dusheiko G, Gilson R, Maini M. FRATRICIDE OF HBV-SPECIFIC CD8 T CELLS BY NK CELLS
195 MEDIATED THROUGH THE TRAIL PATHWAY. JOURNAL OF HEPATOLOGY. 54: S40- S41. Mar 2011 (oral presentation EASL 2011)
11. Nebbia G, Peppa D, Schurich A, Khanna P, Singh H, Rosenberg W, Dusheiko G, Gilson R, Chinaleong J, Kennedy P, et al. Role of the TIM-3/Galectin-9 pathway in chronic hepatitis B infection. IMMUNOLOGY. 135: 69-69. Dec 2011
12. Peppa D, Gill U, Micco L, Schurich A, Nebbia G, Singh H, Rosenberg W, Gilson R, Kennedy PT, Maini M. Susceptibility of T cells to death ligand-mediated deletion in the liver of chronic hepatitis B virus (CHB) infected patients. IMMUNOLOGY. 135: 60-60. Dec 2011
13. Gill U, Micco L, Li L, Peppa D, Ushiro-Lumb I, Foster GR, Maini MK, Kennedy PTF. PEGYLATED INTERFERON A MODULATES INNATE IMMUNITY IN EAG POSITIVE CHRONIC HEPATITIS B AND DETERMINES CHANGES IN HBSAG QUANTIFICATION. Annual Meeting on British-Society-of-Gastroenterology, Birmingham, ENGLAND, 14 Mar 2011 - 17 Mar 2011. GUT. B M J PUBLISHING GROUP. 60: A232-A232. Apr 2011
14. Micco L, Peppa D, Loggi E, Panno AM, Shurich A, Cursaro C, Bihl F, Bernardi M, Andreone P, Maini MK. DIFFERENTIAL EFFECTS OF PEGYLATED INTERFERON ALPHA THERAPY ON INNATE AND ADAPTIVE IMMUNE RESPONSES IN CHRONIC HEPATITIS B. 45th Annual Meeting of the European-Association-for-the-Study-of-Liver-, Vienna, AUSTRIA, 14 Apr 2010 - 18 Apr 2010. JOURNAL OF HEPATOLOGY. ELSEVIER SCIENCE BV. 52: S5-S5. Jan 2010
15. Dimitra Peppa, Claire Dunn, Celeste Pallant, Abhishek Das, Pooja Khanna, Geoffrey Dusheiko, Richard Gilson, Mala K Maini. The cytokine milieu impairs NK cell antiviral capacity in chronic HBV infection. 2009 International Meeting The Molecular Biology of Hepatitis B Viruses, Tours, France (Oral Presentation)
16. Das A, Ellis G, Blair P, Brunetta M, Lopes AR, Khanna P, Dusheiko G, Peppa D, Mauri C, Maini M. Role of IL-10-producing regulatory B-cells in chronic HBV infection. Annual Congress of the British-Society-of-Immunology, Glasgow, SCOTLAND, 17 Nov 2008 - 21 Nov 2008. IMMUNOLOGY. WILEY-BLACKWELL PUBLISHING, INC. 125: 121-122. Dec 2008
17. Peppa D, Dunn C, Pallant C, Das A, Ellis G, Gilson R, Maini M. IMPAIRED INNATE IMMUNE RESPONSES IN PATIENTS WITH CHRONIC HBV INFECTION. J INFECTION 57(5):428-429 Nov 2008
196
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223 Published December 17, 2012 Article
Up-regulation of a death receptor renders antiviral T cells susceptible to NK cell–mediated deletion
Dimitra Peppa,1,2 Upkar S. Gill,3 Gary Reynolds,4 Nicholas J.W. Easom,1 Laura J. Pallett,1 Anna Schurich,1 Lorenzo Micco,1 Gaia Nebbia,1 Harsimran D. Singh,1 David H. Adams,4 Patrick T.F. Kennedy,3 and Mala K. Maini1
1Division of Infection and Immunity and 2Centre for Sexual Health and HIV Research, University College London, London NW3 2PF, UK 3Centre for Digestive Diseases, Institute of Cell and Molecular Science, Barts and the London SMD, London E1 2AD, UK 4Centre for Liver Research and NIHR Biomedical Research Unit in Liver Disease, University of Birmingham, Birmingham B15 2TT,
England, UK Downloaded from
Antiviral T cell responses in hepatotropic viral infections such as hepatitis B virus (HBV) are profoundly diminished and prone to apoptotic deletion. In this study, we investigate whether the large population of activated NK cells in the human liver contributes to this process. We show that in vitro removal of NK cells augments circulating CD8+ T cell
responses directed against HBV, but not against well-controlled viruses, in patients with jem.rupress.org chronic hepatitis B (CHB). We !nd that NK cells can rapidly eliminate HBV-speci!c T cells in a contact-dependent manner. CD8+ T cells in the liver microcirculation are visualized making intimate contact with NK cells, which are the main intrahepatic lymphocytes expressing TNF-related apoptosis-inducing ligand (TRAIL) in CHB. High-level expression of the TRAIL death receptor TRAIL-R2 is found to be a hallmark of T cells exposed to the milieu of the HBV-infected liver in patients with active disease. Up-regulation of TRAIL-R2 on December 18, 2012 renders T cells susceptible to caspase-8–mediated apoptosis, from which they can be par- tially rescued by blockade of this death receptor pathway. Our !ndings demonstrate that NK cells can negatively regulate antiviral immunity in chronic HBV infection and illustrate a novel mechanism of T cell tolerance in the human liver.
CORRESPONDENCE T cell responses are tightly regulated to maintain (Vivier et al., 2008). Accumulating data high- Mala K. Maini: immune homeostasis and limit damage to vital light the capacity of NK cells to also exert a [email protected] The Journal of Experimental Medicine organs. T cells in the liver, in particular, are sub- negative regulatory e"ect on T cells (Su et al., Abbreviations used: CHB, jected to potent tolerizing mechanisms. Although 2001) through inhibition of antigen presen- chronic hepatitis B; CMV, these mechanisms prevent overzealous responses tation (Andrews et al., 2010), production of IL-10 cytomegalovirus; EBV, Epstein– causing tissue injury, they may be exploited by (Lee et al., 2009), or direct killing of T cells. Barr virus; HBV, hepatitis B virus; LCMV, lymphocytic hepatotropic pathogens to subvert antiviral im- Several receptor–ligand interactions between NK choriomeningitis virus; TRAIL, munity (Protzer et al., 2012). There have been cells and T cells have been found to be capable TNF-related apoptosis- major recent advances in our understanding of of leading to autologous lysis of activated T cells inducing ligand. the multiple co-inhibitory pathways driving T cell (Rabinovich et al., 2003; Cerboni et al., 2007; exhaustion in the liver and perpetuating persistent Lu et al., 2007; Soderquest et al., 2011). More viral infections (Protzer et al., 2012). However, the recently, NK cells have been shown to limit potential for NK cells to regulate T cell immunity T cell immunity in a mouse model of chronic has not been de!ned in human viral infections. viral infection (Waggoner et al., 2010; Lang NK cells can contribute to the containment et al., 2012; Waggoner et al., 2012). of many infections by intracellular pathogens (Orange et al., 2002; Khakoo et al., 2004; © 2013 Peppa et al. This article is distributed under the terms of an Attribution– Noncommercial–Share Alike–No Mirror Sites license for the !rst six months after Lodoen and Lanier, 2006; Alter et al., 2011), the publication date (see http://www.rupress.org/terms). After six months it is acting though cytolytic or noncytolytic e"ects on available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/ target cells or by promoting adaptive immunity by-nc-sa/3.0/).
The Rockefeller University Press $30.00 J. Exp. Med. 2013 Cite by DOI: 10.1084/jem.20121172 1 of 16 www.jem.org/cgi/doi/10.1084/jem.20121172 Published December 17, 2012
In this study, we sought to investigate the impact of NK were able to simultaneously compare the e"ect of NK depletion cells on antiviral T cell responses in the setting of persistent on HBV- and CMV-speci!c responses; those directed against an infection with a human hepatotropic virus. Activated NK immunodominant CMV epitope showed a nonsigni!cant trend cells are markedly enriched in the liver microcirculation, to decrease, contrasting with the increase in T cells directed where we hypothesized they would come into prolonged, against HBV in the same patients (Fig. 2 A). Similarly, EBV- and close contact with in!ltrating T cells. Although NK cells in in#uenza-speci!c T cell responses in patients with CHB were patients with chronic hepatitis B (CHB) infection have impaired not augmented upon the removal of NK cells (Fig. 2 B). In pa- noncytolytic antiviral function, we have previously shown tients persistently infected with another hepatotropic virus, hep- that they maintain their cytotoxic potential and up-regulate atitis C (Table S3), there was also no consistent increase in T cells the death ligand TRAIL, particularly in the intrahepatic com- able to recognize 2 immunodominant HLA-A2–restricted HCV partment (Dunn et al., 2007; Peppa et al., 2010). HBV-speci!c epitopes upon NK cell depletion (Fig. 2 C). These data therefore CD8+ T cells, which are essential for viral control, are pro- point to di"erential NK cell regulation of T cells according to foundly depleted in these patients (Maini et al., 2000; Boni their antigen speci!city. et al., 2007). Here, we demonstrate that hepatitis B virus–speci!c T cells up-regulate a death receptor for TRAIL and become NK cells limit HBV-speci!c CD8+ T cell responses susceptible to NK cell–mediated killing, thereby contributing in a contact-dependent manner by inducing apoptosis Downloaded from to the failure of antiviral immunity in CHB. Our initial !ndings suggested NK cells could be suppressing HBV-speci!c CD8+ T cells in culture or could be actively RESULTS deleting them. To investigate the latter possibility, freshly puri- Recovery of HBV-speci!c CD8+ T cells !ed NK cells were re-added at day 10, just 6 h before analysis after depletion of NK cells of HBV-speci!c CD8+ T cell numbers. A diminished HBV- To investigate whether NK cells have the potential to regulate speci!c CD8+ T cell response was observed irrespective of +
virus-speci!c CD8 T cells, we initially determined the impact the timing of NK cell re-addition (representative example jem.rupress.org of total NK cell depletion on the magnitude of HBV-speci!c and summary data can be found in Fig. 3 A). The speed of this T cell responses. CD8+ T cell responses against a pool of peptides e"ect precluded a requirement for the inhibition of prolifera- representing well-described HLA-A2–restricted HBV epitopes tive expansion of virus-speci!c CD8+ T cells in culture, instead or overlapping peptides (15mers) spanning the core protein of indicating that these populations were amenable to rapid HBV were identi!ed by IFN-G production after short-term cul- functional inactivation or deletion by NK cells.
ture. Fig. 1 A is a representative example of HBV responses from To distinguish between these two possibilities, HBV- on December 18, 2012 a patient with active CHB in the presence or absence of NK speci!c CD8+ T cells were identi!ed by HLA-A2/peptide cells. Stimulation of whole PBMCs resulted in the expected low multimer rather than IFN-G staining after removal of NK cells frequency of responses, in line with the well-established paucity at day 0 or 1 and re-addition of a physiological ratio of freshly of detectable HBV-speci!c T cells in CHB (Maini et al., 2000; isolated NK cells on day 0 or 10. Multimer-binding CD8+ Boni et al., 2007). Upon NK cell depletion, there was an en- T cells were only increased if NK cells were removed at the be- hancement of HBV-speci!c CD8+ T cells, which returned to ginning of the culture (not on day 1), and were decreased again baseline levels after re-addition of puri!ed NK cells at a physio- after re-addition of NK cells on day 0 or 10 (Fig. 3 B). These logical ratio at the start of culture. Individual responses and sum- results indicated that NK cells were able to rapidly deplete the mary data are depicted in Fig. 1 (B and C), showing a signi!cant number of virus-speci!c CD8+ T cells surviving in culture. recovery of HBV-speci!c CD8+ T cells upon NK cell depletion The mechanism of action of NK cells on virus-speci!c from patients with CHB. To exclude any potential contribution T cell survival during short-term culture was further investigated of other lymphocyte subsets, including NKT cells, depletion ex- by transwell experiments. A physiological ratio of NK cells was periments were also performed after #ow-cytometric sorting of re-added at day 0 directly to the culture or to transwells separated NK cells to 99% purity (Fig. 1 D). Removal of NK cells also from the T cells by a semipermeable membrane. When NK cells promoted the expansion of a population of CD8+ T cells able to were not in contact with T cells, their survival was enhanced to a bind HLA-A2/HBV peptide multimers (Fig. 1 E). This implied degree similar to that seen upon NK cell depletion (Fig. 3 C, top). that NK cells were in#uencing the number of HBV-speci!c The degree of pancaspase activation in HBV-speci!c T cells (in- CD8+ T cells surviving in culture, not just the function of pre- dicating apoptosis induction) was reduced when NK cells were existing populations. depleted or not in contact with T cells, whereas re-addition of NK cells increased the amount of early (FLICA+7AAD) and Differential effects of NK cells late (FLICA+7AAD+) apoptotic events (Fig. 3, C and D, and not according to T cell speci!city depicted). Thus, NK cell induction of T cell apoptosis required To explore whether NK cell depletion could similarly augment cell–cell contact. other virus-speci!c responses, we analyzed CD8+ T cells directed Collectively, these results indicate that at least some of the against immunodominant HLA-A2–restricted epitopes from cy- impact of NK cells on HBV-speci!c T cells is mediated by a di- tomegalovirus (CMV), Epstein–Barr virus (EBV), and in#uenza rect e"ect on their survival that is contact-dependent and results in our cohort of CHB patients. In 22 patients with CHB, we in caspase activation.
2 of 16 NK cells kill antiviral T cells | Peppa et al. Published December 17, 2012 Article Downloaded from jem.rupress.org on December 18, 2012
Figure 1. Recovery of HBV-speci!c CD8+ T cells after depletion of NK cells. (A) Representative FACS plots from a CHB patient. HBV-speci!c CD8+ T cells were identi!ed by intracellular cytokine staining for IFN-G after 10-d stimulation with a pool of HBV peptides of PBMCs or PBMCs depleted of NK cells ($NK). Where indicated, a physiological ratio of NK cells was re-added in the culture at day 0 before stimulation. (B) Individual responses of CHB patients (n = 27) and matched summary data (C). (D) NK cells were sorted to 99% purity by "ow cytometry. Control PBMCs stained with the same antibodies were passed though the machine untouched. Summary bar graph of (n = 3) experiments. (E) HBV-speci!c CD8+ T cells were identi!ed by MHC peptide multimer staining after short-term peptide stimulation in the absence or presence of NK cells or NK cell re-addition at day 0. Representative FACS plots from a CHB patient and summary data from (n = 9) CHB patients. Error bars represent the mean ± SEM. ND = not detected. ***, P < 0.001; **, P < 0.01.
JEM 3 of 16 Published December 17, 2012
Increased TRAIL-R2 expression on T cells in CHB receptor pathway from the TNF superfamily that was able Several ligand–receptor interactions could be responsible to induce apoptosis through caspase 8 (Sprick et al., 2000). for mediating NK cell killing of T cells through caspase in- We focused on the TRAIL pathway because we have pre- duction. We noted that HBV-speci!c CD8+ T cells stained viously found TRAIL to be up-regulated on NK cells dur- directly ex vivo had an increase in their expression of acti- ing episodes of HBV-related liver inflammation (Dunn vated caspase 8 compared with CMV-speci!c or global CD8+ et al., 2007; Peppa et al., 2010). We therefore investigated T cells (unpublished data). This indicated a death ligand whether the dysregulated T cell response in patients with Downloaded from jem.rupress.org on December 18, 2012
Figure 2. Differential regulation by NK cells according to T cell speci!city. (A) Representative FACS plots for CMV-speci!c responses from a CHB patient upon PBMC stimulation and in the absence of NK cells ($NK) and summary bar graphs comparing CD8+ T cell responses upon HBV or CMV pep- tide stimulation within the same (n = 22) CHB patients ± NK cell depletion. (B) Representative FACS plots for EBV and in"uenza virus–speci!c responses from a CHB patient upon PBMC stimulation and in the absence of NK cells ($NK) and summary data for EBV (n = 6) and in"uenza (n = 7). (C) Example of HCV virus–speci!c CD8+ T cell identi!cation by tetramer staining during short-term culture in the presence and absence of NK cells and summary bar graph from n = 8 HCV patients. Error bars represent the mean ± SEM. **, P < 0.01.
4 of 16 NK cells kill antiviral T cells | Peppa et al. Published December 17, 2012 Article Downloaded from jem.rupress.org on December 18, 2012
Figure 3. NK cells limit the survival of CD8+ HBV-speci!c T cells in a contact-dependent manner by inducing apoptosis. (A) Representative FACS plots from a CHB patient. HBV-speci!c CD8+ T cells were identi!ed by intracellular cytokine staining for IFN-G after 10-d stimulation with a pool of HBV peptides upon NK cell depletion ($NK) or re-addition of freshly puri!ed NK cells (physiological ratio) at day 0 or 10. Summary data from (n = 4) CHB patients. (B) FACS plots from a CHB patient depicting HBV-speci!c CD8+ T cells identi!ed by multimer staining, after depletion of NK cells at day 0, or from PBMC culture at 24 h (Day 1) and re-addition of freshly puri!ed NK cells at a physiological ratio on day 0 or 10. Summary bar graphs of n = 5 experiments. (C, top) Representative FACS plots from a CHB patient. HBV-speci!c CD8+ T cells were identi!ed by multimer staining after short-term peptide stimulation in the absence ($NK) or presence of NK cells. NK cells, where indicated, were re-added at a physiological ratio directly in the culture or were plated into transwells to prevent contact. (bottom) The corresponding proportions of apoptotic virus-speci!c cells. The degree of pancaspase activation was determined by "ow cytometry using the carboxy"uorescein-FLICA apoptosis detection kit. Histograms represent early apoptotic (FLICA+7AAD) and late apoptotic (FLICA+7AAD+) virus-speci!c CD8+ T cells. (D) Summary stacked bars of n = 3 experiments.
JEM 5 of 16 Published December 17, 2012
CHB might become susceptible to TRAIL-mediated kill- infection (Dunn et al., 2007) and is known to have higher ing by up-regulation of TRAIL death receptors. TRAIL affinity for membrane-bound TRAIL (Schneider et al., binding to the death receptors TRAIL-R1 and TRAIL-R2, 1998; Ichikawa et al., 2001). We therefore compared the which contain intracytoplasmic domains, triggers caspase expression of TRAIL-R2 on global T cells in CHB and activation and induction of apoptosis (Schneider et al., 1997; healthy controls directly ex vivo. Levels of TRAIL-R2 Kimberley and Screaton, 2004). TRAIL-R2 is up-regulated were low on peripheral CD8+ T cells in CHB but were on the surface of hepatocytes during active #ares of HBV signi!cantly elevated compared with the negligible levels Downloaded from jem.rupress.org on December 18, 2012
Figure 4. Higher levels of TRAIL-R2 on T cells in patients with CHB. (A) Representative FACS plots and isotype control from a healthy individual and a CHB patient showing expression of TRAIL-R2 on global peripheral CD8+ T cells, and summary data from n = 18 healthy and n = 27 CHB patients. (B) FACS plots depicting identi!cation of virus-speci!c CD8+ T cells ex vivo via multimer staining from a representative CHB patient, and gating strategy showing ex- pression of TRAIL-R2 on virus-speci!c cells. A control multimer was used to help identify the virus-speci!c populations. (C) Paired data showing expression of TRAIL-R2 on HBV versus CMV virus-speci!c CD8+ T cells from (n = 9) CHB patients. (D) Representative FACS plot from an individual with resolved HBV infec- tion, showing gating for HBV-speci!c CD8+ T cells and TRAIL-R2 expression. (E) Summary bar graphs of TRAIL-R2 expression on HBV-speci!c CD8+ T cells from seven individuals with resolved HBV infection and nine CHB individuals. Error bars represent the mean ± SEM. *, P < 0.05; **, P < 0.01.
6 of 16 NK cells kill antiviral T cells | Peppa et al. Published December 17, 2012 Article
seen in healthy controls (Fig. 4 A). No signi!cant corre- TRAIL-R2 expression was then compared on CD8+ lation was observed between the levels of expression of T cells directed against CMV or HBV within the same CHB TRAIL-R2 and any virological or clinical parameters in patients, identi!ed directly ex vivo by MHC/peptide multi- the periphery (unpublished data). mer staining (Fig. 4 B). A signi!cantly higher proportion of Downloaded from jem.rupress.org on December 18, 2012
Figure 5. Intrahepatic CD8+ T cells in CHB patients have up-regulated expression of TRAIL-R2. (A) Representative example of immunostaining of paraf!n-embedded liver tissue (derived from a CHB patient undergoing diagnostic biopsy) showing CD8+ T cells and TRAIL-R2 colocalization by immuno- "uorescence. Bars, 20 µm. (B) Representative FACS plots from a CHB patient showing ex vivo expression of TRAIL-R2 on global peripheral and intrahepatic CD8+ T cells. (C) Ex vivo TRAIL-R2 expression on paired peripheral and intrahepatic global CD8+ T cells from (n = 21) patients with CHB. (D) Correlation of TRAIL-R2 expression on intrahepatic CD8+ T cells and HBV viral load. Spearman r = 0.4870, P value (two-tailed) = 0.02 (E) Comparison of TRAIL-R2 expression on intrahepatic CD8+ T cells from 8 patients with nonviral hepatitis (control), 7 HCV-infected patients, and 21 CHB patients. (F) Co-staining for TRAIL-R2 and HLADR gated on intrahepatic global CD8+ T cells directly ex vivo from a CHB patient, a HCV patient, and a nonviral hepatitis control patient; and summary data from control (n = 5), HCV (n = 6), and CHB (n = 10) patients. ***, P < 0.001; **, P < 0.01; *, P < 0.05.
JEM 7 of 16 Published December 17, 2012 Downloaded from jem.rupress.org on December 18, 2012
Figure 6. TRAIL-R2 expression is a feature of CD8+ T cells encountering antigen in the HBV-infected liver. (A) Representative gating strategy identifying intrahepatic TRAIL-R2+ high (red), TRAIL-R2+ low (black), and TRAIL-R2 negative (gray) CD8+ T cells from a CHB patient. Histograms and bars depict the proportions of CD38 (n = 4), Annexin V (n = 3), PD1 (n = 6), and CD57 (n = 8) expressed by each subset directly ex vivo. (B) The maturation status of intrahepatic TRAIL-R2+ high (red), TRAIL-R2+ low (black), and TRAIL-R2 negative (gray) CD8+ T cells were analyzed by co-staining for CD27 and CD45RA. Representative FACS plot and summary bars of frequencies of expression of TRAIL-R2+ high (red), TRAIL-R2+ low (black), and TRAIL-R2 negative (gray) CD8+ T cells with naive, central memory (CM), effector memory (EM) and revertant (EMRA) phenotypes from n = 4 CHB patients. (C) Summary bar
8 of 16 NK cells kill antiviral T cells | Peppa et al. Published December 17, 2012 Article
HBV-speci!c CD8+ T cells expressed TRAIL-R2 compared To further explore the characteristics of global intrahe- with CMV-speci!c responses (Fig. 4 C), in line with their patic CD8+ T cells bearing TRAIL-R2, we compared the differential susceptibility to NK cell modulation. No up- expression of a panel of phenotypic markers on TRAIL-R2 regulation of TRAIL-R2 was observed on HBV-speci!c CD8+ high, low, and negative fractions (Fig. 6, A and B). TRAIL-R2 T cells obtained from individuals who had previously resolved expressing CD8+ T cells were enriched for the activation HBV infection (Fig. 4, D and E). These data indicated that marker CD38 (in line with the co-staining with HLA-DR TRAIL-R2 was preferentially induced on HBV-speci!c demonstrated in Fig. 5 F) and the apoptosis marker Annexin V, CD8+ T cells encountering their cognate antigen. but not for the exhaustion marker PD-1 (Fig. 6 A). Cells expressing high levels of TRAIL-R2 were less likely to be Further up-regulation of TRAIL-R2 found in the CD57+ and CD27-CD45RA+ subsets and were on intrahepatic CD8+ T cells in CHB instead mainly of a central memory phenotype (Fig. 6, A and B). Because the TRAIL-R2 death receptor was up-regulated in These data suggest that TRAIL-R2–expressing CD8+ T cells CHB, particularly on HBV-speci!c T cells, we hypothesized are activated, apoptotic responses rather than exhausted or that its expression might be further enriched in the liver, the senescent populations. site of HBV replication. To determine whether TRAIL-R2 The large proportion of total intrahepatic CD8+ T cells was expressed on CD8+ T cells in!ltrating the liver, we ini- bearing TRAIL-R2 in CHB led us to question whether its Downloaded from tially examined para$n-embedded sections of HBV-infected expression was restricted to HBV-speci!c T cells. In !ve livers by immuno#uorescence. Using this approach, we noted HLA-A2+ individuals from whom su$cient liver-in!ltrating clear co-staining of CD8+ T cells in the liver with TRAIL-R2 lymphocytes could be obtained, responses directed against (Fig. 5 A). We expanded on these data by extracting liver- HBV or CMV were compared directly ex vivo by HLA-A2/ in!ltrating lymphocytes from surplus liver biopsy tissue, allow- peptide multimer staining. The expression of TRAIL-R2 was ing #ow cytometric comparison of TRAIL-R2 expression on higher on intrahepatic CMV-speci!c CD8+ T cells compared + +
paired circulating and intrahepatic CD8 T cell samples. In with the low levels seen on CD8 T cells of this speci!city in jem.rupress.org 19 out of 21 patients with CHB, the expression of TRAIL- the periphery. However, TRAIL-R2 was further enriched on R2 on CD8+ T cells was further increased in the liver com- intrahepatic HBV-speci!c CD8+ T cells (Fig. 6 C). Upon rec- pared with the peripheral compartment (Fig. 5 B, C). There ognition of their cognate antigen after overnight stimulation was considerable variability in TRAIL-R2 expression levels with HBV peptides, the majority of virus-speci!c CD8 from on intrahepatic CD8+ T cells (Fig. 5 C), and this was found to HBV-infected livers expressed TRAIL-R2 (Fig. 6 D). An up-
correlate positively with HBV viral load (Fig. 5 D). These data regulation of TRAIL-R2 was also observed after stimulation on December 18, 2012 suggested that the liver environment and HBV may both be with CMV peptide on intrahepatic virus-specific CD8+ factors driving up-regulation of this death receptor. To address T cells from HBV-infected patients (Fig. 6 D). In contrast, this, we obtained surplus liver tissue from seven patients with CMV-speci!c CD8+ T cells from control livers without hep- HCV and eight individuals without viral hepatitis. Liver- atitis failed to express any TRAIL-R2 upon peptide stimula- in!ltrating CD8+ T cells from controls without viral hepatitis tion (Fig. 6 D). Thus, high TRAIL-R2 expression appeared to expressed more TRAIL-R2 than peripheral CD8+ T cells be a hallmark of T cells encountering their cognate antigen in from healthy donors, but much less than those from HBV- the milieu of an HBV-infected liver. infected livers (Fig. 5 E). CD8+ T cells extracted from HCV- infected livers showed a level of TRAIL-R2 expression that was TRAIL blocking partially recovers HBV-speci!c CD8+ T cells intermediate between control and HBV-infected livers (Fig. 5 E). We next sought functional evidence for the involvement of Levels of TRAIL-R2 were lower on CD8+ T cells from HCV- the TRAIL pathway in NK-cell mediated deletion of virus- infected as opposed to HBV-infected livers, despite the for- speci!c T cells. To do this, we examined the impact of TRAIL mer group tending to be more in#amed (based on ALT levels; blockade at the time of peptide stimulation of PBMCs on Tables S1 and S2). Levels of TRAIL-R2 were not simply a re- HBV-speci!c CD8+ T cell responses in vitro. TRAIL block- #ection of the degree of T cell activation; signi!cantly more ing experiments were performed with a TRAIL-R2 Fc, com- TRAIL-R2 was coexpressed by the HLA-DR+ (activated) pared with a control IgG1 Fc to exclude a nonspeci!c e"ect fraction of CD8+ T cells extracted from HBV-infected livers (Fig. 7 A). Fig. 7 A presents representative FACS plots from a compared with HCV or control livers (Fig. 5 F). CHB patient and summary data from nine CHB patients
graphs comparing expression of TRAIL-R2 on global intrahepatic CD8+ T cells, CMV-speci!c and HBV-speci!c CD8+ T cells. Virus-speci!c CD8+ T cells were identi!ed directly ex vivo by multimer staining in (n = 5) HLA-A2+ CHB patients with available liver biopsies. (D) Representative examples of the gating strategy from a control patient with no evidence of viral hepatitis (control liver) and a CHB patient (HBV liver) showing expression of TRAIL-R2 on intrahepatic global CD8+ T cells and virus-speci!c CD8+ T cells identi!ed via IFN-G staining after overnight stimulation with CMV peptide and HBV overlapping peptides (OLP), respectively. Summary bar graphs comparing expression of TRAIL-R2 on CMV-speci!c CD8+ T cells from control livers (n = 5) versus CMV and HBV-speci!c CD8+ T cells from (n = 5) CHB patients. Bars represent the mean ± SEM. *, P < 0.05.
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during short-term culture, which shows enhanced responses PBMCs depleted of NK cells did not show any further in- upon TRAIL blockade. However, the e"ect of TRAIL block- crease in the responses rescued above that observed upon NK ade was less striking than that of NK cell depletion in the depletion alone (Fig. 7 A). same patients (Fig. 7 A), suggesting that NK cells were using No signi!cant recovery of CMV-speci!c CD8+ T cells additional pathways for deleting T cells. Although T cells can from the same patients with CHB was observed upon TRAIL express TRAIL in some circumstances (Mirandola et al., 2004; blockade (Fig. 7 B), in keeping with the low levels of TRAIL- Janssen et al., 2005), in our cohort, TRAIL expression was R2 expressed on these cells in the circulation and the lack of con!ned to NK cells (unpublished data), and we could there- their reconstitution upon NK cell depletion. Further support fore assume that TRAIL blockade of PBMCs was acting on for the preferential deletion of a selective subset of TRAIL- NK cells. In support of this approach, TRAIL blocking of R2-bearing T cells by NK cells came from the !nding that Downloaded from jem.rupress.org on December 18, 2012
Figure 7. Partial recovery HBV-speci!c CD8+ T cells to TRAIL blockade. (A) Representative FACS plots from a CHB patient after short-term peptide stimulation of PBMCs in thepresence or absence of NK cells and in the presence of TRAIL-R2/Fc blocking or IgG1-Fc control. Plotted are summary paired data from (n = 9) CHB patients. (B) Representative FACS plot and summary data from n = 6 CHB patients showing the effect of TRAIL-R2 Fc addition at the time of PMBC stimulation with CMV peptide during short-term culture. (C) Representative example from a CHB patient demonstrating the expression of TRAIL-R2 on global CD8+ T cells in the presence or absence ($NK) of NK cells after short-term culture with HBV peptides and summary data from (n = 19) CHB patients. Bars represent the mean ± SEM ***, P < 0.001; **, P < 0.01; *, P < 0.05.
10 of 16 NK cells kill antiviral T cells | Peppa et al. Published December 17, 2012 Article
the percentage of global CD8+ T cells expressing TRAIL-R2 To test whether the T cells in!ltrating HBV-infected liv- was increased after depletion of NK cells from HBV peptide- ers were subject to death receptor-mediated apoptosis, we stimulated PBMC cultures (Fig. 7 C). stained them for caspase 8, which is activated by these path- ways from the TNF superfamily (Sprick et al., 2000). Caspase 8 was detectable in a proportion of CD8+ T cells isolated Overnight rescue of intrahepatic HBV-speci!c from HBV-infected liver biopsies; its expression was enriched T cells by TRAIL blockade in the expanded population of intrahepatic CD8+ T cells NK cells constitute up to 40% of the lymphocytic in!ltrate expressing TRAIL-R2 on their surface (Fig. 8 B), in line with within the liver, the site of HBV replication. We have previ- their enhanced expression of Annexin V (Fig. 6 A). These ously shown that intrahepatic NK cells in patients with HBV- !ndings support the capacity of TRAIL-R2 to deliver an related in#ammation are highly activated and express increased apoptotic signal to the large proportion of intrahepatic CD8+ levels of TRAIL compared with circulating NK cells, whereas T cells on which it is expressed in CHB. intrahepatic CD3+ T cells express little TRAIL (Dunn et al., To assess whether TRAIL blocking was capable of restoring 2007). The extensive sinusoidal network in the liver forms a virus-speci!c T cell responses in the liver, we examined paired unique vascular bed with a narrow lumen and sluggish blood samples from nine HLA-A2 CHB patients from whom suf- !cient liver biopsy tissue was available for functional experi-
#ow. We postulated that this would facilitate close associations Downloaded from between NK cells and T cells as they in!ltrated the liver. To ments. Overlapping peptides (15mers) spanning the core investigate this, we performed staining of para$n-embedded protein of HBV were used for overnight stimulation, and liver sections from patients with CHB by immunohistochem- both peripheral and intrahepatic virus-speci!c T cell re- istry. Using this approach we were able to visualize intimate sponses were assessed for IFN-G production in the presence contact between NK cells and T cells in the HBV-infected or absence of TRAIL blocking. Despite the fact that many liver sinusoids, as exemplified in Fig. 8 A. This supported of these cells were already poised to die, with activation +
the concept that the intrahepatic predominance of TRAIL- of intracellular caspases, intrahepatic HBV-speci!c CD8 jem.rupress.org expressing NK cells, in conjunction with the unique archi- T cell responses could be augmented after just overnight tecture of the liver encouraging close contact between TRAIL blockade in four out of nine patients (Fig. 8 C). In lymphocyte subsets, would promote the deletion of TRAIL- contrast, the expansion of detectable HBV-speci!c T cell R2–bearing T cells. responses from PBMCs could only be achieved after 10 d on December 18, 2012
Figure 8. Overnight recovery of intrahe- patic HBV-speci!c T cells by TRAIL block- ade. (A) Examples of NK cells (NKp46 in blue) in intimate contact with CD3+ T cells (red) in the sinusoidal spaces of a representative HBV- infected liver. Immunohistochemistry was performed on paraf!n-embedded HBV tissue. Bars 20 µm. (B) Representative FACS plot from a CHB patient and gating strategy showing expression of caspase 8 in intrahepatic TRAIL- R2+ CD8+ T cells ex vivo and summary bar graphs comparing expression of caspase 8 in global CD8+ T cells versus TRAIL-R2+ CD8+ T cells (n = 5). Only live events were analyzed. (C) Representative FACS plots showing re- sponse to TRAIL blockade after overnight incubation of intrahepatic cells with overlap- ping (OLP) HBV peptides and individual intra- hepatic responses from nine CHB patients with available liver tissue.
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rather than overnight TRAIL blockade (unpublished data). over-ridden according to the type of infection. This concept is These results underscore the susceptibility of the enriched popu- supported by population genetic studies that have demonstrated lation of HBV-speci!c T cells in the liver compartment to NK that KIR haplotypes are under balancing selection and can be cell TRAIL-mediated deletion. associated with resistance to infection (Khakoo et al., 2004; Alter et al., 2011) but also autoimmunity and immunopathology DISCUSSION (Parham, 2005; Kulkarni et al., 2008). Although speculative, it is Persistent viral infection with HBV is associated with several possible that reported associations between KIR haplotypes T cell–intrinsic and –extrinsic defects, culminating in pro- and outcome of infections with HIV, HCV and HTLV-1 found depletion of the HBV-speci!c CD8+ T cell responses (Khakoo et al., 2004; Alter et al., 2011; Seich Al Basatena et al., that constitute a critical component of antiviral defense (Boni 2011) may in part be determined by di"erential NK cell regula- et al., 2007; Lopes et al., 2008; Protzer et al., 2012). In this tion of antiviral T cell responses. In CHB, the protective e"ect of study, we addressed the role of NK cells, one of the main MHC class I molecule expression may be insu$cient for self e"ectors of the innate immune response, in HBV-speci!c tolerance in the face of selective up-regulation of receptors/ T cell modulation. Our results demonstrate that NK cells can ligands superseding control of self-killing. limit virus-speci!c T cell responses, contributing to the apop- Although we have delineated a role for the TRAIL path- totic predilection of these cells. NK cells from patients with way, our data imply that additional receptor–ligand interac- Downloaded from CHB can mediate these e"ects without prior activation and tions could contribute to driving NK cell lysis of T cells in at physiological ratios. NK cell–mediated regulation of T cell CHB. The capacity of NK cells to lyse LCMV-speci!c CD8+ responses was found to be partly mediated through TRAIL. T cells has been shown to be regulated by 2B4 (Waggoner We have previously found TRAIL to be up-regulated on NK et al., 2010) or NKG2D (Lang et al., 2012); the impact of HBV cells in #ares of HBV-related liver in#ammation and enriched infection on relevant pathways regulating NK cell cytotoxic- in the intrahepatic compartment (Dunn et al., 2007). Here, ity merits investigation. The recent studies in LCMV show + +
we show that HBV-speci!c CD8 T cells, particularly those that NK cells can regulate CD8 T cell responses not only by jem.rupress.org in!ltrating the liver, up-regulate the TRAIL-R2 death- direct killing (Waggoner et al., 2010; Lang et al., 2012), as inducing receptor. Therefore, NK cell–mediated deletion of demonstrated in our experiments, but also via elimination virus-speci!c T cells via TRAIL may perpetuate viral persis- of CD4+ T cell responses (Waggoner et al., 2012). This !n d - tence and immunopathology, especially within the in#amma- ing may also be pertinent to the liver, where CD4+ T cell tory milieu of the intrahepatic compartment. numbers are already low and priming is defective (Wuensch
There is accumulating evidence suggesting that NK et al., 2010). on December 18, 2012 cells have an important immunoregulatory role (Zingoni The contrasting e"ects of NK cell removal on HBV- et al., 2004; Martín-Fontecha et al., 2004; Morandi et al., speci!c T cells and responses to unrelated viruses (CMV, 2006; Andrews et al., 2010), in addition to a direct antiviral EBV, and in#uenza) within the same patients pointed to- function in the setting of the early response to infection ward di"erential NK cell–T cell receptor–ligand interac- (Vivier et al., 2008). Our data are the !rst to reveal a path- tions depending on their antigenic exposure. In line with way for direct NK cell regulation of T cells in a human this, TRAIL-R2 was found to be substantially higher on the persistent viral infection. They are supported by recent surface of HBV-speci!c cells compared with CMV-speci!c studies highlighting a detrimental role of NK cells on the cells, suggesting that the features of HBV-speci!c CD8+ antiviral response in a murine model of persistent viral in- T cells that target them for NK cell–mediated deletion are not fection (Waggoner et al., 2010, 2012; Lang et al., 2012). shared by T cells of all virus speci!cities. The expression of Activated NK cells have been described to target activated TRAIL-R2 was increased globally on activated, apoptotic T cells in a cytolytic manner, resulting in a signi!cant loss intrahepatic CD8+ T cells, although more so in CHB than of lymphocytic choriomeningitis virus (LCMV)–speci!c HCV-infected or other control liver samples. It is conceiv- CD8+ T e"ector cells, which leads to impaired virus control able that antigen presentation in the tolerogenic liver and altered immunopathology (Waggoner et al., 2010; Lang environment is able to impose a TRAIL-R2-expressing et al., 2012). A follow-up study further highlighted a role phenotype on T cells, as described for the pro-apoptotic for NK cells as rheostats, regulating CD4+ T cell–mediated molecule Bim (Holz et al., 2008). The hepatic cytokine mi- support of CD8+ T cells, and thereby controlling LCMV patho- lieu (Dunn et al., 2007) and HBV antigens (Janssen et al., genesis and persistence (Waggoner et al., 2012). Thus, de- 2003) can up-regulate TRAIL-R2 expression on hepato- pending on the context of infection and viral kinetics, cytes, and could likewise a"ect its expression on intrahepatic NK cells may dictate viral clearance or immunopathology. T cells. In addition, reactive oxygen species (ROS) have NK cell activation is the result of the balance of signals been shown to regulate T cell apoptosis (Hildeman et al., from a complex array of activatory and inhibitory receptors, 1999) and influence the expression of TRAIL receptors combined with the cytokine milieu. The fact that NK cells (Kwon et al., 2008) and may represent an important mecha- can potentially have either a bene!cial or deleterious e"ect nism in the liver. on autologous T cells implies that the protective e"ect of Although healthy T cells are normally protected against MHC in maintaining self-tolerance may be supported or the apoptotic e"ects of soluble TRAIL (Mirandola et al., 2004),
12 of 16 NK cells kill antiviral T cells | Peppa et al. Published December 17, 2012 Article
several studies suggest that viral infections can render MATERIALS AND METHODS lymphoid cells susceptible to TRAIL-mediated cytotoxic- Patients and healthy controls. Patients were recruited from Mortimer ity (Katsikis et al., 1997; Jeremias et al., 1998; Miura et al., Market Clinic (London), the Royal Free Hospital (London), and the Royal London Hospital. Full ethical approval was obtained and each patient gave 2001). We !nd that cells expressing high levels of TRAIL- written informed consent. All CHB patients were anti-HCV and anti-HIV R2 in the liver are poised to die, with elevated levels of in- antibody negative and treatment naive. Seven patients who had resolved tracellular caspase 8. We would therefore anticipate that previous HBV infection and 18 age-matched healthy volunteers donated receptor blockade would be more e"ective in vivo than in blood for the study. Patient characteristics are included in Table I. Liver vitro, allowing for rescue of newly generated antiviral samples were obtained from 21 HLAA2 patients with CHB and paired pe- CD8+ T cells before they are driven to their apoptotic fate. ripheral and intrahepatic samples. Surplus liver tissue was available from !ve HLAA2+ and four HLA-A2 patients with CHB undergoing diagnostic We have previously demonstrated that pretreatment of liver biopsies (Table S1). Characteristics of patients with HCV and controls PBMCs from HBV patients with a pancaspase inhibitor without viral hepatitis from whom liver tissue was available are outlined in before peptide stimulation can rescue HBV-specific CD8+ Table S2. Characteristics of HCV patients from whom only PBMCs were T cells that have up-regulated Bim (Lopes et al., 2008). used shown in Table S3. TRAIL signaling can also lead to Bim up-reglation (Han PBMC and intrahepatic lymphocyte isolation. PBMCs were isolated et al., 2006; Cummins and Badley, 2009) and it is likely that by gradient centrifugation on Ficoll-Hypaque and frozen or immediately
the two pathways converge (Bouillet and O’Reilly, 2009), studied as described later. Sera were collected and frozen for later use. Intra- Downloaded from increasing the apoptotic propensity of virus-speci!c cells. hepatic lymphocytes were isolated as previously described (Peppa et al., TRAIL expression on NK cells is increased during disease 2010) In brief, liver tissue was suspended in RPMI-1640 (Sigma-Aldrich) #ares (Dunn et al., 2007), which would be predicted to result in and macerated with a plunger from a 25-ml syringe and a scalpel in a Petri an accelerated deletion of T cells expressing TRAIL receptors. dish. The cell suspension was then passed several times through a 70-mm cell strainer (BD), washed three times, and resuspended in RPMI complete me- Ongoing high-level expression of TRAIL-R2–bearing T cells dium with 10% fetal bovine serum for counting. Lymphocytes were identi- in the HBV-infected liver is consistent with the constant !ed under a high magni!cation by their size, shape, and granularity.
renewal of antigen-primed T cells in persistent viral infec- jem.rupress.org tions (Vezys et al., 2006) and with our phenotypic character- Puri!cation of NK cells. Freshly puri!ed NK cells from PBMCs of CHB ization of these populations. patients were isolated (>96% purity and viability; NK isolation kit; Miltenyi In the in#amed HBV-infected liver, the prominence Biotec) as per the manufacturer’s instructions. NK cells were depleted by CD56 MACS microbeads (Miltenyi Biotec); TRAIL expression on NK of activated NK cells bearing high levels of TRAIL may cell–enriched fractions was con!rmed by #ow cytometry. Where indicated, have the dual e"ect of both contributing to death of he- separated NK cells were plated into transwells (1 µm pore size; Polycarbon-
patocytes (Dunn et al., 2007) and of promoting apoptosis ated Membrane; Corning Costar). FACS of NK cells (99% purities) was per- on December 18, 2012 of T cells with up-regulated TRAIL receptors. However formed on the basis of CD56 expression (CD56+CD3) by FACSAria (BD). it is important to note that TRAIL-bearing NK cells have Control PBMCs stained with the same antibodies were passed though the machine untouched. also been suggested to protect against tumors in the murine liver (Takeda et al., 2001) and to counteract liver !brosis Flow cytometric analysis. For phenotypic analysis, PBMCs isolated from in chronic hepatitis C (Glässner et al., 2012). With these HBV patients and healthy donors were washed in PBS, and surface stained considerations in mind, the timing of TRAIL blockade in at 4°C for 20 min with saturating concentrations of monoclonal anti-CD3 the liver would need to be carefully considered. As a short- PE-Cy7, CD8 Alexa Fluor 700, HLADR V500, CD19 V450, CD4 APC- term adjuvant to the immunotherapy of CHB, TRAIL block- Cy7 (eBioscience), CD56-TEXAS Red (Beckman Coulter), and TRAIL- R2 (R&D Systems) in the presence of !xable live/dead stain (Invitrogen). ade could promote antiviral responses while minimizing Where stated, the degree of activated caspase 8 and activated pancaspases liver in#ammation. were determined using the FAM-LETD-FMK or the carboxy#uorescein- In summary, we provide evidence of a novel pathway (FAM-VAD-FMK) FLICA kit (Serotec) according to the manufacturer’s in- whereby activated NK cells may excessively down-modulate structions. For further phenotypic analysis of intrahepatic CD8+ T cells, the the antiviral immune response in CHB. NK cell–mediated following antibodies or isotype-matched controls were used: CD38-FITC deletion of T cells may represent an important homeostatic (BD), CD45RA V450 (eBioscience), CD27 V500 (BD), PD1-PERCP (eBioscience), CD57-APC (BD) in the presence of !xable live/dead stain control mechanism to prevent exuberant T cell responses in (Invitrogen). Where indicated, the viability of CD8+ T cells was further the liver that has been hijacked by HBV to assist it in its on- assessed by staining for Annexin V (BioLegend) according to the manu- going battle to evade immune control. facturer’s protocol in the presence of 7AAD viability staining solution.
Table 1. Characteristics of patients from whom PBMCs alone were available Age in years Sex (female:male) HBeAg+ HBV DNA IU/ml ALT IU/L: median median (range) median (range) (range) CHB patients (n = 27) 34 (23-51) 4:23 3/27 1,500 (36–7.3 × 107) 38 (17–378) Resolved HBV (n = 7) 39 (29-69) 1:6 NA NA NA Healthy controls (n = 18) 32 (21-49) 6:12 NA NA NA NA, not applicable.
JEM 13 of 16 Published December 17, 2012
The frequencies of HBV peptide-speci!c cells from HLA-A2+ individuals Immunohistochemistry and immuno"uorescence. Sections of archival were evaluated directly ex vivo or after short-term culture by multimer para$n-embedded HBV tissues were dewaxed and rehydrated, and epitope staining as previously described. In brief, total PBMCs were stained with retrieval was performed as previously described (Dunn et al., 2007). All washes APC-labeled HBV c18-27, envelope 183–191, envelope 335–343, envelope for immunostaining techniques and dilution of antibodies were done with 348–357, and polymerase 508–510 dextramers (Immudex) at 37°C for EnVision FLEX wash bu"er (Dako). After 2 min in Harris hematoxylin 15 min in complete RPMI plus 10% FCS. The cells were then pelleted and to block auto#uorescence, immunostaining was performed on a Shandon stained as above. A control dextramer was used to identify the population of Sequenza. After a 10-min endogenous protein block in 2% Casein solution positive cells. For the analysis of HCV-speci!c CD8+ T cells PE-labeled (Vector), primary antibodies mouse anti-CD8 (Vector) at 1/50 dilution and HLA-A2–restricted MHC class I tetrameric complexes speci!c for HCV polyclonal rabbit anti-DR5 (Abcam) at 1/100 were applied for 1 h. Visualiza- NS3 1406–1415 (KLSGLGINAV) and HCV NS3 1435–1443 (CVNGVC- tion was performed with anti–mouse DyLight 488 and anti–rabbit DyLight WTV) were used (a gift from E. Barnes, Oxford University, Oxford, Eng- 594 (Vector) at 1/200 dilution for 15 min. Sections were mounted with land, UK). Tetramer staining was considered positive if a distinct population VECTASHIELD, with DAPI (Vector), and captured on a Zeiss Axiovision mi- (>0.02%) could be discriminated. Cells were acquired on a LSRII (BD) and croscope with 1,000× magni!cation. Immunohistochemistry was performed analyzed using FlowJo (Tree Star). on a Dako autostainer. After a 10-min peroxidase block (Dako), a 10-min en- dogenous protein block in 2% Casein solution was applied. Sections were in- Peptide stimulation. PBMCs or PBMCs depleted of NK cells were stimu- cubated with mouse anti-CD3 (Dako) at 1/100 dilution for 30 min, visualized lated with the following peptides representing HLA-A2–restricted viral epi- with mouse ImmPRESS (Vector), 30 min and ImmPACT NovaRED (Vector) topes: HBV envelope epitopes: FLLTRILTI, WLSLLVPFV, LLVPFVQWFV, for 5 min. After a water wash, 2% Casein solution was reapplied. Sections were
and GLSPTVWLSV; HBV core epitope: FLPSDFFPSV; HBV polymerase epi- then incubated in goat polyclonal anti-NKp46 (R&D Systems) at 1/100 dilu- Downloaded from topes: GLSRYVARL, KLHLYSHPI); CMV pp65 immunodominant epitope: tion for 1 h, followed by biotinylated anti–goat secondary (Vector) and strept- NLVPMVATV; EBV BMLF1 immunodominant epitope: GLCTLVAML and ABC alkaline phosphatase (Vector), as per manufacturer’s recommendations. in#uenza MP 58–66 immunodominant epitope: GILGFVFTL0 (Proimmune). Visualization was completed with Vector blue for 20 min. After counterstaining For stimulation of HLA-A2 patients, a pool of 15mer peptides overlapping with Mayer’s hematoxylin (Leica), slides were rapidly dehydrated, cleared, and by 10 residues spanning the core protein of HBV genotype D (JPT Peptide mounted in VectaMount (Vector Systems). Images were captured on a Leica Techonologies) was used. For control viral responses in HLA-A2 patients, DM with Nikon Coolpix camera at 1,260× magni!cation. CMV peptides spanning the pp65 protein were used (JPT Peptide Technolo-
gies). PBMCs from HCV-infected patients were stimulated with HCV pep- Statistical analysis. Statistical signi!cance was performed between paired jem.rupress.org tides. Amino acid sequences of the speci!c antigenic HCV peptides were samples using the Wilcoxon signed rank test and between HBV patients and identical to those of the respective MHC class I tetrameric complexes used healthy controls using the Mann-Whitney U test. The nonparametric Spear- (provided by E. Barnes). Peptides were dissolved in sterile endotoxin-free man test was used for correlation analysis. P < 0.05 was considered to be sig- DMSO. Final DMSO concentration during culture was <0.1%. ni!cant for all tests.
Short-term culture. Where indicated, PBMCs or PBMCs depleted of NK Online supplemental material. Tables S1–S3 detail clinical characteris-
cells were stimulated with 1 mM peptide (or a pool of the seven HBV peptides) tics of patients from whom liver biopsies or HCV-speci!c responses were on December 18, 2012 in the presence of 20 IU IL-2 in RPMI complete medium for 10 d at 37°C. acquired. Online supplemental material is available at http://www.jem IL-2 and medium were refreshed on day 4 of culture. In selected experiments, .org/cgi/content/full/jem.20121172/DC1. NK cells were depleted from PBMC culture at 24 h (day 1). On day 9, PBMCs were restimulated with 1 mM peptide overnight in the presence of Brefeldin A We thank all the patients and staff who generously helped with the study, and (added 1 h into the incubation). In selected experiments, a physiological ratio Prof. Ian Weller for his invaluable mentoring. (based on the patient-speci!c circulating NK cell frequency) of freshly isolated This work was funded by the Medical Research Council (Clinical Research Training Fellowship to DP and grant G0801213 to M.K. Maini). NK cells was re-added in the culture either at the onset of stimulation or at day M.K. Maini !led a patent (PCT/GB2008/000811) for the use of TRAIL blocking 10. Where indicated, isolated NK cells were plated into transwells (1 µm pore agents in viral hepatitis. The authors have no additional !nancial interests. size; Polycarbonated Membrane; Corning Costar) at onset of culture. Virus- speci!c T cells were identi!ed either via dextramer staining as previously de- Submitted: 1 June 2012 scribed or via ICS for IFN-G. In brief, cells were surface stained, !xed, and Accepted: 20 November 2012 permeabilized, followed by intracellular staining for IFN-G APC (BD). To ex- amine the e"ect of blocking TRAIL on virus-speci!c CD8+ T cells, 1 µg/ml REFERENCES of TRAIL-R2/Fc (R&D Systems), or IgG1-Fc (R&D) control antibody was Alter, G., D. Heckerman, A. Schneidewind, L. Fadda, C.M. Kadie, J.M. added with peptide at onset of culture, and cells were treated as described Carlson, C. Oniangue-Ndza, M. Martin, B. Li, S.I. Khakoo, et al. above. TRAIL-R2/Fc is a dimeric receptor that binds TRAIL ligand with 2011. HIV-1 adaptation to NK-cell-mediated immune pressure. Nature. higher a$nity than the natural receptor, thereby blocking this interaction. The 476:96–100. http://dx.doi.org/10.1038/nature10237 degree of pancaspase activation was determined using the carboxy#uorescein- Andrews, D.M., M.J. Estcourt, C.E. Andoniou, M.E. Wikstrom, A. Khong, (FAM-VAD-FMK) FLICA apoptosis detection kit (Serotec) according to the V. Voigt, P. Fleming, H. Tabarias, G.R. Hill, R.G. van der Most, manufacturer’s protocol for detection by #ow cytometry. FLICA reagent was et al. 2010. Innate immunity de!nes the capacity of antiviral T cells to limit persistent infection. J. Exp. Med. 207:1333–1343. http://dx.doi added at day 10 of culture, for 1 h before staining. PBMCs from HCV-infected .org/10.1084/jem.20091193 patients were stimulated with 1 mM pooled peptides and expanded as above. Boni, C., P. Fisicaro, C. Valdatta, B. Amadei, P. Di Vincenzo, T. HCV-speci!c T cells were identi!ed via tetramer staining as described above. Giuberti, D. Laccabue, A. Zerbini, A. Cavalli, G. Missale, et al. 2007. Characterization of hepatitis B virus (HBV)-speci!c T-cell dysfunc- Overnight stimulation. For overnight stimulation of PBMCs or IHL, tion in chronic HBV infection. J. Virol. 81:4215–4225. http://dx.doi 10 mM peptide was added for 12 h, and the cells were incubated at 37°C in .org/10.1128/JVI.02844-06 the presence of Brefeldin A (added 1 h into the incubation). To examine the Bouillet, P., and L.A. O’Reilly. 2009. CD95, BIM and T cell homeostasis. e"ect of TRAIL blocking these experiments were repeated in the presence Nat. Rev. Immunol. 9:514–519. http://dx.doi.org/10.1038/nri2570 of a combination of TRAIL-R2/Fc and TRAIL-R1/Fc chimeras (R&D Cerboni, C., A. Zingoni, M. Cippitelli, M. Piccoli, L. Frati, and A. Santoni. Systems) added at the time of peptide stimulation. Virus-speci!c T cells were 2007. Antigen-activated human T lymphocytes express cell-surface identi!ed by ICS for IFN-G. NKG2D ligands via an ATM/ATR-dependent mechanism and become
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16 of 16 NK cells kill antiviral T cells | Peppa et al. Blockade of Immunosuppressive Cytokines Restores NK Cell Antiviral Function in Chronic Hepatitis B Virus Infection
Dimitra Peppa1,2, Lorenzo Micco1, Alia Javaid3, Patrick T. F. Kennedy3, Anna Schurich1, Claire Dunn1, Celeste Pallant1, Gidon Ellis1, Pooja Khanna1,4, Geoffrey Dusheiko4, Richard J. Gilson2, Mala K. Maini1,2,3* 1 Division of Infection and Immunity, UCL, London, United Kingdom, 2 Centre for Sexual Health and HIV Research, UCL, London, United Kingdom, 3 Centre for Digestive Disease, Barts and The London School of Medicine and Dentistry, London, United Kingdom, 4 Centre for Hepatology, Hampstead Campus, Royal Free & University College Medical School, London, United Kingdom
Abstract NK cells are enriched in the liver, constituting around a third of intrahepatic lymphocytes. We have previously demonstrated that they upregulate the death ligand TRAIL in patients with chronic hepatitis B virus infection (CHB), allowing them to kill hepatocytes bearing TRAIL receptors. In this study we investigated whether, in addition to their pathogenic role, NK cells have antiviral potential in CHB. We characterised NK cell subsets and effector function in 64 patients with CHB compared to 31 healthy controls. We found that, in contrast to their upregulated TRAIL expression and maintenance of cytolytic function, NK cells had a markedly impaired capacity to produce IFN-c in CHB. This functional dichotomy of NK cells could be recapitulated in vitro by exposure to the immunosuppressive cytokine IL-10, which was induced in patients with active CHB. IL-10 selectively suppressed NK cell IFN-c production without altering cytotoxicity or death ligand expression. Potent antiviral therapy reduced TRAIL-expressing CD56bright NK cells, consistent with the reduction in liver inflammation it induced; however, it was not able to normalise IL-10 levels or the capacity of NK cells to produce the antiviral cytokine IFN-c. Blockade of IL-10 +/2 TGF-b restored the capacity of NK cells from both the periphery and liver of patients with CHB to produce IFN-c, thereby enhancing their non-cytolytic antiviral capacity. In conclusion, NK cells may be driven to a state of partial functional tolerance by the immunosuppressive cytokine environment in CHB. Their defective capacity to produce the antiviral cytokine IFN-c persists in patients on antiviral therapy but can be corrected in vitro by IL-10+/2 TGF-b blockade.
Citation: Peppa D, Micco L, Javaid A, Kennedy PTF, Schurich A, et al. (2010) Blockade of Immunosuppressive Cytokines Restores NK Cell Antiviral Function in Chronic Hepatitis B Virus Infection. PLoS Pathog 6(12): e1001227. doi:10.1371/journal.ppat.1001227 Editor: Luca G. Guidotti, The Scripps Research Institute, United States of America Received July 30, 2010; Accepted November 11, 2010; Published December 16, 2010 Copyright: ß 2010 Peppa et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was funded by Medical Research Council Awards G108515 and G0801213 to MKM, a CRDC Fellowship to DP and an EASL Fellowship to LM. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected]
Introduction the delicate balance between protective and pathogenic responses. IFN-c can clear HBV-infected hepatocytes through non-cytolytic NK cells constitute a major cellular arm of the innate immune mechanisms[7,8]. NK cell-derived IFN-c could therefore consti- system and, as such, have been viewed as most relevant in the tute a vital antiviral mechanism in the liver, where hepatocytes are setting of the initial response to an acute infection. However, they relatively resistant to the cytolytic mechanisms of perforin and may also be appropriately or inappropriately activated to exert granzyme production[9]. effector function when persistent infection and its pathological The intensity and quality of NK cell effector function is sequelae become established. Their role may be particularly determined by the balance of activatory and inhibitory signals important in patients with CHB, in whom the virus-specific CD8 through their array of receptors (NK-R), in addition to the T cell arm of protection is markedly diminished and dysfunctional influences exerted by the cytokine microenvironment. The TRAIL [1,2]. pathway of NK cell-mediated hepatocyte killing can be driven by NK cells are greatly enriched in the liver, the site of HBV the cytokines IFN-a and IL-8, induced during flares of CHB[4]. replication[3,4]. We have previously demonstrated an increase in Similarly, NK cells in HCV infection can be polarised towards activated CD56bright NK cells in the livers of patients undergoing cytolysis and expression of TRAIL as a result of exposure to flares of eAg-negative CHB. This subset can be induced to express endogenous[10] or therapeutic[11] IFN-a. Conversely, intrahe- TNF-related apoptosis-inducing ligand (TRAIL), which is able to patic NK cell function can be down-regulated by the immuno- kill hepatocytes that have upregulated death-inducing TRAIL suppressive cytokine IL-10 produced by Kupffer cells[12]. In receptors, thereby contributing to liver inflammation in CHB[4]. addition, a role for IL-17 in curtailing NK cell function was The CD56bright subset can also be a potent source of cytokines recently demonstrated in disseminated vaccinia virus infection of such as IFN-c[5,6], a key cytokine shaping adaptive immunity and mice with pre-existing dermatitis[13]. In this study we have
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Author Summary lymphocytes in these patients (Fig 1f), this corresponds to a substantial enrichment of CD56bright NK cells in the liver. Hepatitis B virus (HBV) infection is responsible for more than a million deaths annually as a result of the immune- Impaired non-cytolytic antiviral potential of NK cells in mediated chronic liver damage it induces. One of the key CHB immune players in the liver is the natural killer (NK) cell, We have previously shown that the CD56bright subset of NK which we have recently found can cause liver damage in cells can mediate hepatocyte apoptosis through expression of the HBV infection. Here we address the antiviral potential of NK cells in the HBV-infected liver and demonstrate that death ligand TRAIL in flares of eAg-negative CHB[4]. In this cohort of patients we confirmed an increase in TRAIL expression they have a specific impairment in their ability to produce bright the cytokine IFN-c, which could limit their capacity to (largely on the CD56 subset, Fig 2a representative plots) in control HBV. We find that the potent antiviral drugs patients with either eAg+ or eAg- CHB who had evidence of liver currently being used to treat HBV infection are unable to inflammation (Fig2a summary data). bright fully reverse this NK cell functional defect. We define a role The CD56 subset of NK cells can also be a potent source of for the immunosuppressive cytokine environment in HBV IFN-c[14], a cytokine that has direct non-cytolytic antiviral effects in down-regulating NK cell antiviral function, which can be on HBV replication [7,8] and can promote adaptive immune restored by specific blockade of IL-10 and TGF-b. This work responses[6]. Despite the enrichment of CD56bright NK cells in therefore highlights a mechanism contributing to the CHB, we found that they had an impaired capacity to produce failure of immune control in chronic HBV infection, paving IFN-c (representative plots, Fig2b). There was a significant the way to new therapeutic options. reduction in production of IFN-c by NK cells from 46 patients with CHB compared to 29 healthy controls (Fig2b). This reduction was seen irrespective of disease activity (liver inflammation Fig2b, investigated cytokine-driven modulation of IFN-c production by viral load or eAg status, data not shown) or method of NK cell NK cells in patients with CHB and explored the potential to stimulation (IL-12/IL-18 (Fig2b), IL-12/IL-15, K562 with IL-12/ restore their non-cytolytic antiviral function. IL-18 or PMA/ionomycin, data not shown). Both the CD56bright subset and the CD56dim subset (that has recently been recognised Results to also make a contribution to cytokine production[15]) showed c bright significantly impaired IFN- production (FigS1a). Similarly, Expansion of the CD56 subset of NK cells in CHB CD56bright and CD56dim NK cells in CHB showed a trend to To explore NK cell effector potential in the setting of persis- produce less TNF-a, despite the strong stimulus required to tent HBV infection, we first analysed the frequency of reliably elicit this cytokine (FigS1b). Simultaneous assessment of bright dim neg dim pos CD56 (CD16 / ) and CD56 (CD16 ) NK cell subsets IFN-c and TNF-a production showed a significant reduction in in 64 patients with CHB compared to 31 healthy age-matched dual producing NK cells in CHB (FigS1c). bright controls (Table 1). The proportion of circulating CD56 NK To assess NK cell cytolytic potential, we determined their cells was significantly increased in patients with CHB (represen- capacity to degranulate as evidenced by CD107 expression tative FACS plots Fig1a, summary data Fig1b), with a tendency to following stimulation with K562 target cells and cytokines. There further increases in those with liver inflammation (Fig1b). There was no significant difference in NK cell degranulation potential in was a trend for the percent of circulating NK cells to decrease in 33 patients with CHB compared to 21 controls (Fig2c). Differential CHB (Fig 1c) but the absolute number of circulating CD56bright analysis by NK cell subset or by patient disease status did not show NK cells was still significantly increased (p,0.05 data not any differences (data not shown). NK cells in CHB were therefore shown). biased towards cytolytic and death-ligand mediated effector To determine whether there was a further enrichment of this functions and defective IFN-c production. immunoregulatory CD56bright NK cell subset at the site of viral To determine the potential of potent antiviral treatment to replication, we compared the proportions in intrahepatic and correct this bias in NK cell effector function, we studied a group of circulating lymphocytes. In all eight patients with CHB from 22 patients with HBV viraemia well-suppressed on a combination whom paired samples were available, the percent of CD56bright of of Lamivudine and Adefovir. Upon viral suppression and total NK cells was higher in the intrahepatic compared to normalisation of liver inflammatory markers, there was no peripheral compartment (Fig1d,e). Since NK cells make up a significant change in the percent of NK cells (FigS2a), but the significantly greater proportion of intrahepatic than circulating proportion of CD56bright NK cells decreased to levels observed in
Table 1. Characteristics of study population.
Treatment Group HBV Patients HBV Patients (Lamivudine Healthy Controls High ALT Low ALT and Adefovir) n=31 n=29 n=35 n=22
Age, years: median (range) 30 (18–52) 43.5 (23–65) 32 (23–65) 43 (18–70) Sex (Female:male) 14:17 14:15 16:19 5:17 ALT IU/L: median (range) na 112 (57–604) 34 (10–47) 25 (18–70) HBV DNA IU/mL: median (range) na 1,546,000 (1150–2.96108) 870 (100–3.36108) ,100 HBeAg+ na 18 3 6
na = not applicable. doi:10.1371/journal.ppat.1001227.t001
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Figure 1. NK cell frequency and altered subset distribution in the periphery and intrahepatic compartment. (A) Representative density plots gated on CD3- PBMC and co-stained for CD56 and CD16 to identify NK cells from a healthy control and a CHB patient. (B) Summary data of the proportions of CD56bright subset in the periphery of CHB patients with low ALT (n = 35, ALT ,50IU/L, median 34) compared to high ALT (n = 29, median ALT 112) and healthy controls (n = 31). (C) Frequency of circulating NK cells in CHB patients with low ALT and high ALT and healthy controls. (D) Density plots of NK cells from peripheral blood and intrahepatic lymphocytes from a representative CHB patient. (E) Paired cumulative results of peripheral and intrahepatic CD56bright NK cells frequencies from 8 patients with CHB. (F) NK cell frequency in peripheral blood and intrahepatic compartment from 8 patients with CHB with paired samples. The non-parametric Mann-Whitney U test was used to compare data between groups and the Wilcoxon signed rank test was used between paired variables. *p,0.05 or ** p,0.01 designates values that differ significantly between groups. Ctr = healthy controls. doi:10.1371/journal.ppat.1001227.g001 healthy controls (Fig2d); in line with this, NK cell TRAIL activity on NK cells is mediated indirectly via other constituents expression reduced to baseline levels (Fig2d). However NK cell such as APCs. The contrasting effects of IL-10 on TRAIL and IFN-c production was only partially augmented upon antiviral IFN-c expression represented differential regulation of these treatment (mainly CD56dim subset, FigS2b) and remained effector functions in the same NK cells rather than the emergence significantly lower than that in healthy controls (Fig2d). of two distinct subsets. The small population of TRAIL-expressing NK cells present in healthy donors were at least as able to produce IL-10 is induced in CHB and recapitulates the NK cell IFN-c as the rest of the NK cell population (FigS3c). The addition defect in IFN-c production of exogenous IL-10 suppressed IFN-c in NK cells regardless of Effector function of NK cells is tightly regulated by the cytokine their TRAIL expression (FigS3c). In line with this, gating on the milieu and their production of IFN-c can be inhibited by expanded population of TRAIL-expressing NK cells found in immunosuppressive cytokines such as IL-10[12,16] and IL- CHB demonstrated that their IFN-c-producing capacity was no 17[13]. The levels of IL-17A were not elevated in sera from more reduced than that of the non-TRAIL-expressing fraction patients with CHB compared to controls (Fig3a). In contrast, (FigS3d). circulating concentrations of IL-10 were significantly increased in patients with active HBV disease (Fig3b,c by CBA, confirmed by Restoration of NK cell IFN-c production upon blockade of ELISA, data not shown), correlating with viral load (r = 0.48, immunosuppressive cytokines p = 0.002) and ALT (r = 0.37, p = 0.03). IL-10 levels showed a Since IL-10 was induced in CHB and exogenous IL-10 was able trend to decrease on antiviral treatment but remained significantly to mimic the selective suppression of NK cell effector function, we higher than in controls (Fig3c), consistent with the limited next investigated the potential to restore NK cell IFN-c production restoration of NK cell IFN-c production in these patients. by IL-10 blockade. Addition of antiIL10/IL10-R blocking mAbs To test whether IL-10 could induce the defect in NK cell IFN-c restored the ability of both CD56bright and CD56dim NK cells from production seen in CHB, we re-assessed NK cell effector function patients with active CHB to produce IFN-c (mean 2.5 fold with or without the addition of exogenous IL-10. IL-10 increase, Fig4a,b,d). The majority of patients without biochemical significantly suppressed NK-cell derived IFN-c (Fig3d), particu- evidence of liver inflammation (and with low viral loads) did not larly in those patients in whom it was not already substantially respond to this strategy (Fig4c,d), in line with their lower levels of reduced (Fig3e, and in healthy controls, data not shown). By circulating IL-10 (Fig3b). A subset of those patients failing to contrast, IL-10 had no effect on cytolytic ability or TRAIL respond to IL-10 blockade did show recovery of NK cell IFN-c phenotype (Fig3f) and did not affect the percent of NK cells production following blockade of both IL-10 and TGFb, another (FigS3a). The ability of IFN-a to further induce NK cell TRAIL immunosuppressive cytokine known to be able to inhibit NK cell expression in vitro[4] was also not abrogated by IL-10 (data not production (Fig4e,f). shown). The effect of IL-10 was consistent but more modest on To investigate whether the suppression of NK cell IFN-c was purified NK cells (FigS3b), suggesting that some of its suppressive maintained at the site of HBV replication, paired liver and blood
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Figure 2. Skewed NK cell effector function in CHB is only partially corrected during therapy. (Panels A–C) Representative density plots from a healthy control and HBV patients with low ALT (ALT ,50 IU/L, median 33) and raised ALT (ALT.50 IU/L, median 112) and summary data for TRAIL expression, IFN-c production and CD107 expression. (D) Summary bar charts of CD56bright proportions, NK cell TRAIL expression and NK cell IFN-c production from healthy, CHB and patients on antiviral therapy. Results are expressed as mean 6 SEM. Rx = treated patients. *p,0.05, **p,0.01, ***p,0.001 by Mann-Whitney test. doi:10.1371/journal.ppat.1001227.g002 samples from eight patients with CHB were examined (Table 2). The fold increase in the capacity of CD56bright NK cells to secrete CD56bright NK cell IFN-c production showed a trend to be even IFN-c upon IL-10/TGFb blockade was greater in the liver than lower in the liver than the periphery of patients with CHB the periphery (Fig5a,b). (FigS4a). Levels of intrahepatic NK cell IFN-c production did not significantly correlate with levels of ALT (FigS4b), viral load or Discussion liver histology in this small sample of patients, only one of whom had histological evidence of significant liver inflammation (Table Accumulating evidence points to a contribution of NK cells in 2). Due to limited cell numbers, individual cytokine blockade could the battle to control persistent intracellular pathogens[6,17,18]. not be performed but dual IL-10/TGFbRII blockade reconstitut- Although NK cells have been considered part of the innate ed the proportion of NK cells able to produce IFN-c (%positive, immune response, recent data have suggested that they can possess Fig5a) and increased their level of IFN-c production (MFI, Fig5b). properties previously ascribed to the adaptive arm, including the
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Figure 3. IL-10 is elevated in CHB and suppresses NK cell IFN-c production. (A) Levels of cytokines IL-17A and (B) IL-10 determined using Cytometric Bead Arrays flex sets using sera from 13 healthy controls, 14 low ALT (median ALT 35, all eAg-) and 21 high ALT patients (median ALT 115, 13eAg-). (C) Cumulative IL-10 results including therapy group (n = 13, median ALT 25). (D) Representative density plots of the effect of exogenous IL- 10 on IFN-c production by NK cells from a CHB patient and (E) paired cumulative results from 19 CHB patients. (F) Summary bar charts of the effect of exogenous IL-10 on the expression of TRAIL and CD107 in 5 CHB patients. Results are paired and expressed as mean 6 SEM. Stimulus = IL12+IL18. Significance determined by the Mann-Whitney test for comparison between groups and the Wilcoxon signed rank test for paired data, *p,0.05, **p,0.01, ***p,0.001. doi:10.1371/journal.ppat.1001227.g003 capacity to develop memory and tolerance[19,20,21]. In this study CHB[24]. Instead, our data suggest that the selective NK cell we show that NK cells can develop selective defects in antiviral functional defects seen in this infection may be attributable to the function in the setting of chronic infection and inflammation, immunosuppressive cytokine milieu. reminiscent of the hierarchical loss of effector function manifested Our analysis of NK cell effector potential in a large cohort of by exhausted T cells[22]. patients with CHB revealed preservation of cytolytic capacity and Just as T cell defects have been attributed to excessive antigenic an increase in TRAIL-bearing CD56bright NK cells. Despite this stimulation, functional impairment of NK cells has been ascribed increase in the subset of NK cells that are usually the most potent to excessive stimulatory signals through the activating receptor source of cytokines[14], there was a decrease in the overall NK cell NKG2D, resulting in its down-modulation[19,20]. This is a capacity to produce IFN-c. Such divergence of effector function is plausible mechanism in CHB since data from transgenic mice in line with the recent finding that cytokines are trafficked and suggest that HBV can upregulate the intrahepatic expression of secreted via completely different pathways to cytotoxic granules in NKG2D ligands[23]. However, a recent study and our unpub- NK cells[25]. Consistent with these distinct trafficking pathways, lished data do not support this mechanism, showing no down- separate signalling pathways have been shown to control the regulation of NKG2D or consistent changes in other NK cell release of cytokines and cytotoxic granules in NK cells[26,27]. receptors that could account for the NK cell impairment seen in Unique molecular switches are starting to be identified that couple
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Figure 4. IL-10 blockade alone or in combination with TGFbRII blocking restores NK cell IFN-c production. (A) Representative density plot from a CHB patient of peripheral NK cell IFN-c production in the presence of anti-IL-10 and anti-IL10 receptor blocking mAb. (B) Paired summary data from CHB patients with either active disease (High ALT median 104, n = 13) or (C) inactive disease (Low ALT median 33, n = 9). (D) Fold change in IFN-c produced by total NK cells following IL-10 blockade in both groups of patients. (E, F) Representative density dot plots from a CHB patient and summary bar chart of paired results from 11 patients (n = 11 median ALT 42) of NK cell IFN-c production following IL-10 blockade alone or in combination with anti-TGFbRII blocking antibodies. Stimulus = IL12+IL18. Significance determined by the Mann-Whitney test for comparison between groups and the Wilcoxon signed rank test for paired data, *p,0.05, **p,0.01. doi:10.1371/journal.ppat.1001227.g004
NK cell receptor signalling with the generation of cytokines rather with active HBV infection to produce IFN-c. IL-10 was not able to than cytotoxic functions[28,29]. It is therefore conceivable that a inhibit cytotoxic degranulation and could not overcome the pathway specific to NK cell cytokine production is dysregulated in capacity of IFN-a to induce TRAIL, in line with the maintenance patients with CHB. of these pathogenic functions of NK cells in CHB. IL-10 was The immunosuppressive cytokine IL-10 has been shown to consistently modestly elevated in the serum of patients with CHB, specifically impair NK cell IFN-c production[30], in contrast with but would be expected to be at higher concentrations at the site of IL-17 and excessive NKG2D signalling, both of which result in infection in the liver and in close proximity to the cells from which down-modulation of all NK cell effector functions[13,20]. The it is released. NK cells themselves can produce IL-10[14,32] to liver is an immunotolerant organ, predisposed to the production of allow auto-suppression, but in the HBV-infected liver there are a immunosuppressive cytokines; down-regulation of intrahepatic number of other candidate cellular sources and there is likely to be NK cell IFN-c production has been linked to the local release of a complex regulatory network involved in maintaining its IL-10 by Kupffer cells[12,31]. We found that exposure of NK cells production, as recently described in HIV infection[33]. to IL-10 in vitro was able to recapitulate the selective reduction in We recently reported a transient induction of IL-10 in early IFN-c production noted in patients with CHB. Furthermore, its acute HBV infection that was temporally associated with a blockade was able to restore the capacity of NK cells from patients transient suppression of the capacity of NK cells to produce IFN-c,
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Table 2. Patient characteristics with available liver biopsy specimens.
Age Sex HBV DNA (IU/mL) ALT (IU/L) Necro- Patients Median 35.5 M:F Median 66,879 Median 56 inflammatory Modified ISHAK n=8 Range 24–66 6:2 HBeAg+ 2/6 Range 646–1.26106 Range 15–113 score Stage Fibrosis
Pt1 25 F Pos 113,757 113 2/18 1/6 Pt2 32 M Pos 310,000 63 4/18 3/6 Pt3 49 M Neg 700,000 15 na 1/6 Pt4 40 F Neg 20,000 26 3/18 1/6 Pt5 24 M Neg 947 86 2/18 1/6 Pt6 66 M Neg 646 56 3/18 1/6 Pt7 39 M Neg 6500 26 3/18 1/6 Pt8 27 M Neg 1.26106 56 3/18 1/6
na = not available. doi:10.1371/journal.ppat.1001227.t002 coincident with the increase in viraemia and production of viral proteins in these patients may play a role. In patients with low level antigens[16]. In our cohort of patients with CHB it was difficult to CHB without evidence of liver inflammation, IL-10 was not distinguish the influence of viraemia or liver inflammation, since elevated and its blockade alone could not rescue NK function, both were increased in patients with elevated levels of IL-10. which instead required additional TGF-b blockade. TGF-b is Future study of a group of patients with high viral load but normal another immunosuppressive cytokine that characterises the ALT (immunotolerant phase) could help to dissect the role of these tolerising liver environment and has been shown to be increased factors. The fact that NK cell IFN-c production and IL-10 levels in CHB[34]. TGF-b has been shown to be an alternative key were not significantly normalised by potent antiviral therapy regulator of the capacity of human NK cells to produce IFN-c, suggests that the continued secretion of high levels of HBV suppressing IFN-c and T-bet via Smad2/3/4[35].
Figure 5. Blockade of IL10/TGF enhances intrahepatic NK cell IFN-c production. (A) Representative density plots and (B) histograms for total intrahepatic NK cell and CD56bright subset IFN-c production upon blockade with anti-lL-10, anti-IL10 receptor and anti-TGFbRII blocking antibodies. Paired summary bar charts of fold change increase in the percentage and mean fluorescence intensity (MFI) of NK total and CD56bright IFN-c+ cells in the periphery and intrahepatic compartment of 7 CHB (median ALT 56). Results are expressed as mean 6 SEM. Stimulus = IL12+IL18. *p,0.05 by Wilcoxon signed rank test. doi:10.1371/journal.ppat.1001227.g005
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The collective action of TGF-b and IL-10 may represent an cytometer (Becton Dickinson) and analysed using Flowjo analysis important feedback mechanism to limit exuberant immune software (Treestar). responses and tissue immunopathology in a vital organ like the liver. However, in the context of chronic infections, elevated levels Cytokine production by intracellular staining may attenuate immune responses sufficiently to contribute to the As previously described[16], PBMC were incubated with failure of resolution of infection. A role for IL-10 in persistent viral 50 ng/mL of rhIL-12 (Miltenyi) and rhIL-18 (R&D Systems, infection has been highlighted recently by studies showing that Abingdon, U.K.) for 21 hours at 37uC. 1mM monensin (Sigma- blockade of the IL-10 receptor is associated with resolution of Aldrich, Gillingham, U.K.) was added for the final 3 hours. Cells LCMV infection[36,37]. Genetic studies have also highlighted the were fixed and permeabilised followed by intracellular staining for importance of IL-10 in the antiviral response to HBV; polymor- IFN-c-PE (R&D systems). Where indicated the same experiments phisms of the IL-10 promoter resulting in elevated IL-10 were performed in the presence of rhIL-10 (50ng/mL), or blocking production are associated with viral persistence, increased disease antibodies to anti-IL10 (5 mg/mL) (eBioscience) and anti-IL-10R severity and progression[38,39]. (10 mg/mL) alone or in combination with antiTGFbRII (10 mg/ Our data suggest that immunosupressive cytokines may polarise mL) (BD Biosciences). NK IFN-c production was determined by NK cells in CHB, having no effect on their expression of death subtracting baseline IFN-c production from that observed after c ligands and cytolytic granules but inhibiting IFN- production. cytokine or antibody treatment. NK cells from PBMC of a NK cells expressing death ligands like TRAIL would only be able randomly selected group of patients were isolated (.96% purity to have a direct antiviral effect at the expense of liver damage. The and viability) (Miltenyi Biotec, Germany, NK isolation kit) to decline in liver inflammation seen on antiviral treatment is assess the effect of exogenous IL-10 on IFN-c production. compatible with the reduction in TRAIL-expressing CD56bright For TNF-a production, PBMC were stimulated with phorbol NK cells that we noted in this setting. However, potent antiviral myristate acetate (PMA) (3 ng/mL) and ionomycin (100 ng/mL) therapy was unable to significantly restore the capacity of NK (Sigma) for 3 hours; 1mM monensin (Sigma-Aldrich, Gillingham, cells to produce IFN-c, which would therefore retain an impair- U.K.) was added for the final 2 hours. Cells were then stained with ed capacity for non-cytolytic clearance of HBV from hepatocytes the same antibody combination used for phenotyping prior to and boosting of adaptive immune responses. Our findings raise permealisation and intracellular staining for TNF-a. In selected the possibility of immunotherapeutic targeting of IL-10 and experiments NK cell TNF-a and IFN-c co-expression was assessed TGF-b in CHB, with the caveat that these cytokines govern a critical balance between impeding pathogen clearance and re- following PMA/I stimulation. straining immunopathology. CD107 degranulation assay Materials and Methods As previously described[16], PBMC were incubated with K562 cells (5:1 E:T ratio) for 3 hours at 37uC following overnight Ethics statement stimulation with a combination of rhIL-12/rhIL-18 or medium Clinical assessment and blood sampling were performed during alone in the presence or absence of rh-IL10. CD107a-PE antibody routine hepatitis clinics, with written informed consent and local (BD Biosciences, Cowley, U.K.) was added at the time of ethical board approval of the Royal Free Hospital, the Royal stimulation with target cells and 1mM monensin was added London Hospital and Camden Primary Care Ethics Review during the last two hours of the incubation prior to staining and Board. acquisition.
Patients and healthy subjects Determination of serum cytokine concentrations by All patients were anti-Hepatitis C- and anti-Human Immuno- Cytometric Bead Array (CBA) deficiency Virus-antibody negative and treatment naı¨ve with the CBA flex-sets were used for the determination of IL-10, IL-17 exception of a sub-group of 22 patients suppressed on a (BD Biosciences, Cowley, U.K) according to manufacturers’ combination of Lamivudine and Adefovir. Patient characteristics protocols for serum samples. are included in Table 1. Paired peripheral blood and liver biopsy specimens (surplus to diagnostic requirements) were obtained from Statistical analysis 8 CHB-infected patients (Table 2). Statistical significance was performed between paired samples using the Wilcoxon signed rank test and between HBV patients Isolation and storage of PBMC and Intrahepatic and healthy controls using the Mann-Whitney U test. Correlations lymphocyte isolation between variables were evaluated with the Spearman rank Peripheral blood mononuclear cells (PBMC) were isolated by correlation test. P,0.05 was considered to be significant for all gradient centrifugation on Ficoll-Hypaque and frozen or imme- tests. diately studied as described later. Sera were collected and frozen for later use. Intrahepatic lymphocytes were isolated as previously Supporting Information described[4]. Figure S1 Summary bar charts comparing production of (A) Extracellular staining and flow cytometric analysis IFN-c and (B) TNF-a from NK total and NK cell subsets in For phenotypic analysis, PBMC isolated from HBV patients healthy controls and CHB patients. (C) 10 healthy controls and 12 and healthy donors were stained with fluorochrome-conjugated CHB patients were evaluated for the co-production of TNF-a and antibodies to CD3-Cy5.5/PerCP, CD56-FITC, CD16-APC, and IFN-c following stimulation with PMA/I. Summary bar charts TRAIL-PE or isotype matched controls (BD Biosciences, Cowley, show the percentage of total NK cells that are single positive for U.K.). In selected experiments TRAIL expression was determined IFN-c, TNF-a and double positive for IFN-c/TNF-a.*P,.05, following overnight incubation with 50 ng/mL of rhIL-10 **P,.01, ***P,.001 by Mann-Whitney test. (eBioscience). PBMC were acquired on a FACS Calibur flow Found at: doi:10.1371/journal.ppat.1001227.s001 (0.48 MB EPS)
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Figure S2 (A) Frequencies of circulating NK cells and (B) Figure S4 (A) Production of IFN-c by circulating and intrahe- summary bar chart comparing production of IFN-c from NK total patic NK cells and NK cell subsets from 8 CHB patients with and NK cell subsets in healthy controls (n = 29), CHB patients available liver samples. Paired summary bar charts expressed as (n = 46) and HBV patients on antiviral treatment (n = 20). *P,.05, mean 6 SEM. (B) Lack of significant correlation between **P,.01, ***P,.001 by Mann-Whitney test. intrahepatic NK cell IFN-c production and ALT. Spearman Found at: doi:10.1371/journal.ppat.1001227.s002 (0.46 MB EPS) statistical test was performed (r = -0.39, p = 0.32). Figure S3 (A) Representative FACS plots showing the effect of Found at: doi:10.1371/journal.ppat.1001227.s004 (0.46 MB EPS) exogenous IL10 on NK cells frequencies (boxed CD56+CD32) (B) NK cells from 4 eAg- CHB patients (median ALT 50, median Acknowledgments . VL 2300) were negatively purified ( 96% purities) and stimulated We are grateful to the staff and patients of our clinics for the provision of with IL-12/IL-18 in the presence or absence of exogenous IL-10. the samples used in this study. The effect of IL-10 is shown for the CD56bright subset (**P,.01 significance determined by paired t test). (C) Representative density plots Author Contributions and histograms from a healthy control and (D) a CHB patient showing NK cell IFN-c production, gated on the Conceived and designed the experiments: DP CD CP MKM. Performed CD56+CD32TRAIL- and CD56+CD32TRAIL+ populations, the experiments: DP LM AJ GE. Analyzed the data: DP LM AS MKM. following stimulation with IL12/IL18 +/2 IL-10. NK cell IFN-c Contributed reagents/materials/analysis tools: AJ PTFK PK GD RJG. Wrote the paper: DP MKM. production is expressed as MFI. Found at: doi:10.1371/journal.ppat.1001227.s003 (0.92 MB EPS)
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