Il-7-Mediated Cd56bright Nk Cell Function Is Impaired in Hcv In

IL-7-MEDIATED CD56BRIGHT NK CELL FUNCTION IS IMPAIRED IN HCV IN

PRESENCE AND ABSENCE OF CONTROLLED HIV INFECTION, WHILE

CD14BRIGHTCD16- MONOCYTES NEGATIVELY CORRELATE WITH CD4

MEMORY T CELLS AND HCV DECLINE DURING HCV-HIV CO-INFECTION

CHELSEY J. JUDGE

Submitted in partial fulfillment of the requirements for the degree of

Doctor of Philosophy

Thesis Advisor: Donald Anthony, M.D., Ph.D.

Department of Pathology

CASE WESTERN RESERVE UNIVERSITY

January, 2017 CASE WESTERN RESERVE UNIVERSITY

SCHOOL OF GRADUATE STUDIES

We hereby approve the thesis/dissertation of

Chelsey J. Judge

Candidate for the degree of Doctor of Philosophy*.

Committee Chair

David Canaday

Committee Member

Donald Anthony

Committee Member

Scott Sieg

Committee Member

Daniel Popkin

Committee Member

Clive Hamlin

Date of Defense

November 2, 2016

*We also certify that written approval has been obtained for any proprietary material contained therein TABLE OF CONTENTS

LIST OF TABLES 4

LIST OF FIGURES 5

ACKNOWLEDGMENTS 8

LIST OF ABBREVIATIONS 10

THESIS ABSTRACT 12

Chapter 1. Introduction 14

-Hepatitis C Virus (HCV) and Human Immunodeficiency Virus (HIV) 15

Clinical impact 15

HIV virology overview 17

HIV treatment: Antiretroviral therapy 19

HCV virology overview 20

HCV clinical symptoms and manifestations 25

HCV treatment 26

-Immune Response to HCV 27

Overview of the interplay of the innate and adaptive immune response 27

Natural killer (NK) cells and the potential role for IL-7 on anti-viral effector function 30

NK cell interactions with dendritic cells and monocytes 36

Role of immune activation on the immune response to chronic viral infection 38

Chapter 2. CD56bright16dim/- NK cell IL-7R α-chain (CD127) expression negatively associates with HCV level while IL-7-mediated NK cell cycling and cytolytic function are impaired during HCV and HIV infections 43

-Abstract 44

-Introduction 45

-Methods 47

-Results 54

-Discussion 75

Chapter 3. CD14brightCD16- monocytes and sCD14 level negatively associate with CD4-memory T-cell frequency and predict HCV-decline on therapy 80

-Abstract 81

-Introduction 82

-Methods 83

-Results 87

-Discussion 95

Chapter 4. Summary and Significance of Findings 98

Chapter 5. Future Directions: Determine the role of soluble immune activation mediators on observed IL-7-mediated NK cell function and anti-viral CD4 T-cell response 102 -Characterizing immune activation during HCV therapy 103 Further elucidate the role of soluble immune activation mediators on CD4 memory T-cell activity 104 Determine the effect of soluble immune activation mediators on IL-7-mediated NK cell activity 106 Elucidate the effect of removal of HCV on IL-7-mediated NK cell activity and CD4 memory T-cell frequency and function after DAA therapy 108

-Understand the effect of monocytes on IL-7-mediated NK cell activity and CD4 T-cell anti-viral function 112

-Does IL-7 promote NK-dependent T-cell priming? 114

Determine the potential role of IL-7 on NK editing of DCs, monocytes and macrophages 114

Potential of improving Th1 polarization with IL-7-exposed NK cells 116

Concluding Remarks 118

REFERENCES 120

LIST OF TABLES

Table 2.1. Clinical Parameters of Study Subjects. 48

Supplemental Table 3.1 Clinical Information 86

LIST OF FIGURES

Figure 1.1. Clinical course of HIV infection. 19

Figure 1.2. HCV lifecycle. 24

Figure 1.3. Model. 42

Figure 2.1. CD56bright NK cell CD127 expression negatively associates with HCV level. 55

Supplemental Figure 2.1. CD56bright NK cell CD127 of purified NK cells is unaltered in HCV and HIV infections and negatively associates with HCV level. 56

Figure 2.2. IL-7 activates CD56bright NK cells and enhances IFNα-mediated

CD56bright NK cell activation via CD69 upregulation. 59

Supplemental Figure 2.2. IL-7 upregulates CD56bright NK cell CD69 expression in dose response fashion and promotes CD56bright CD69 upregulation

in HCV infected subjects. 60

Figure 2.3. IL-7 enhances NK cell IFNγ secretion and IFNα-mediated

NK cell IFNγ release. 62

Figure 2.4. IL-7-promotion of CD56bright NK cell Bcl-2 expression and cycling is impaired in HCV and HIV infections. 65

Supplemental Figure 2.3. JFH-1 infected Huh7.5 cell lysis in response to

IL-7 and IFNα. 67

Figure 2.5. IL-7 promotes NK cell cytotoxic function, which is impaired in

HIV and HCV infections. 69

Figure 2.6. IL-7/CD127 signaling is impaired in chronic viral infection. 71

Supplemental Figure 2.4. IL-7-mediated NK IFNγ release is dependent on

AKT and STAT-5. 72

Figure 3.1. CD4CM and EM Cells are reduced in HCV+HIV+ co-infection and negatively correlate with HCV-level and classical monocyte frequency. 89

Supplemental Figure 3.1. Soluble markers of immune activation positively correlate with each other and liver damage score. 91

Figure 3.2. sCD14 is negatively associated with HCV-decline and

CEF-IFNγ response, while CD4EM frequency is positively associated with

HCV-decline and CEF-IFNγ response. 94

Figure 4.1. Plasma IL-6 and sCD14 are elevated in HCV mono-infection. 104

Figure 4.2. Plasma sCD14 and IL-6 levels are increased during

IFN-containing HCV therapy. 110

Figure 4.3. Classical monocytes positively correlate with

CD56-16+ NK cells, while sCD14 negatively associates with

CD56bright NK cells in HCV infection. 113

Acknowledgements

This milestone in my life could never have happened without the support of so many. I am fortunate to have been privileged to earn my PhD at Case Western Reserve

University, in my hometown. CWRU has provided me with access to top professors in the field of immunology and infectious diseases, renowned healthcare facilities and enriching courses. The Immunology Training Program within the Pathology Department created a strong academic environment that allowed students to network with each other and professors. My committee, made up of Drs. Scott Sieg, Dan Popkin, Dave Canaday,

Donald Anthony and Clive Hamlin provided guidance and perspective on my projects and graduate career. Most notably, my thesis advisor Dr. Anthony created a wonderful and warm atmosphere in the lab that motivated me to pursue a career in biomedical research. His encouragement and support pushed me to see my potential in science.

My family and friends have continuously been a source of support and motivation. They have all allowed me to indulge in teaching them a thing or two about the immune system, mainly focused on the importance of their NK cells. When I was over-studied or over- read, they all added levity or an ear for me to vent. My comrades in science, including the combined Anthony/Popkin lab and my science trifecta- Mark, Elane and Luis- challenged me to be a better presenter, writer and researcher. Of course, it was Elane- who listened to every single practice presentation, read over all my writings, and gave me advice based on her experiences- who truly helped shape my approach to science. Without her as my sister in science, this whole PhD process would have been significantly more challenging.

Growing up, I was always told that I could do ‘whatever I put my mind to.’ I never felt pushed in any particular direction or discouraged from pursuing what I was passionate about- my family is awesome that way. Grandma and Larry, you have always been around supporting me and acting as my benefactors. Thank you! My father nicknamed me the brain when I was young, and told me he could see me going to CWRU for a science degree- he has always been a little clairvoyant. He put his money where his mouth was and funded every summer science class I wanted to enroll in, and put me through college. I would not have chosen to pursue a PhD if I had insurmountable college debt. My brother prides himself on ‘making me cool,’ and he’s totally right. Any ounce of cool factor I have is from him, who taught me to not take life so seriously and prioritize fun. Connor has, of course, become an added inspiration to studying the immune system, and I hope that one day I can truly make a contribution to helping him and others with autoimmune diseases. Finally, and most importantly, I would never have finished, or probably even started, this PhD without my mother. She is my number one fan and I live to make her proud, although that is most likely the easiest feat in the world.

She loves unconditionally and believes in who she loves with her whole heart, offering them all the praise and recognition her high-energy being can muster. Because I’m fortunate to be the recipient of such love, I’m able to pursue challenges because I believe in their potential outcome. My mom has acted not only as a mother to me, but an assistant, stylist, financier, fitness and travel partner, friend and sister. I’m sure I’m missing other roles as well. I love you and thank you.

List of Abbreviations

HCV- hepatitis C virus DC- dendritic cell

HIV- human immunodeficiency virus CM- central memory

AIDS- acquired immunodeficiency EM- effector memory syndrome NK- natural killer

ART- antiretroviral therapy NKR- natural killer receptor

HCC- hepatocellular carcinoma KIR- killer immunoglobulin-like

UD- uninfected donor receptor

ACTG- AIDS Clinical Trial Group NCR- natural cytotoxicity receptor

IRB- institutional review board TNF- tumor necrosis factor

DAA- direct acting antivirals TRAIL- TNF-related apoptosis-inducing

AST- aspartate aminotransferase ligand

ALT- alanine aminotransferase FasL- Fas ligand

PLT- platelet JAK- Janus kinase

APRI- AST:platelet ratio index STAT- signal transducers and activators

RBV- ribavirin of transcription

IFN- interferon AKT- protein kinase B

PEG- PEGylated ELISA- enzyme-linked immunosorbent

SVR- sustained virologic response assay

TLR- TOLL-like receptor ELISPOT- enzyme-linked immunospot

Th1- Type 1 T helper MFI- mean fluorescent intensity

PBMC- peripheral blood mononuclear BCL-2- B-cell lymphoma-2 cell IP-10- IFNγ-induced protein-10

10 sCD14- soluble CD14 LPA- lysophosphatidic acid sCD163- soluble CD163 MHC- major histocompatibility complex

IL- interleukin HLA- human leukocyte antigen

CMV- Cytomegalovirus CCR- C-C Motif Chemokine Recepto

CEF- Cytomegalovirus, Epstein-Barr TGF-β- transforming growth factor-beta

Virus and Influenza Virus PD-1- programmed death-1

ENPP2- ectonucleotide PDL- programmed death ligand-1 pyrophosphatase/phosphodiesterase 2 APC- antigen presenting cell

ATX- autotaxin

IL-7-mediated CD56bright NK Cell Function is Impaired in HCV in Presence and Absence

of Controlled HIV Infection, While CD14brightCD16- Monocytes Negatively Correlate

with CD4 Memory T cells and HCV Decline During HCV-HIV Co-infection

Abstract

by CHELSEY J. JUDGE

HCV-infection affects approximately 170 million worldwide, with 60-85 percent of acute-infections resulting in chronic-infection, and a significant proportion are co- infected with HIV. HCV-infection causes increased morbidity and mortality in HCV-HIV co-infection. HIV-infection alters HCV-disease pathogenesis, accelerating progression to cirrhosis and liver failure. While HCV-therapy has greatly improved, the cost remains a barrier and is not expected to completely remove HCV from the population. We need a better understanding of the immune-response to HCV to develop successful vaccine strategies.

HCV-containment and -clearance is dependent on efforts of the innate and adaptive immune-response. CD4 memory T-cells are critical in viral-clearance, by producing IFNγ and mediating CD8 T-cell responses. NK cells have been shown to exert an important role in control of HCV-infection by lysis of infected-cells and cytokine release.

Monocytes may partly shape this HCV-directed response, via direct-contact with CD4 T- cells and NK cells and in production of soluble factors.

IL-7 has been demonstrated to enhance NK cell IFNγ production, and the IL-7 receptor α chain (CD127) is expressed on NK cells. We measured CD127 expression on NK cell subsets of uninfected donors, chronic HCV-infected treatment-naïve, HIV-infected on

ART and HCV-HIV co-infected subjects on ART. We demonstrate that CD56bright NK cell CD127 expression negatively correlated with HCV plasma levels in HCV mono- infection and HCV-HIV co-infection. We observed IL-7-induced NK cell activation, cell- cycling, IFNγ release and cytolytic function, with impairments in HCV- and HIV- infections. These findings offer a role for IL-7-dependent NK cell function in control of chronic viral infections.

Within chronic HCV- and HIV-infection, there have been observations of elevated levels of IL-6 and sCD14, produced in part by monocytes, which contribute to immune- activation. We extended these studies to evaluate the role of immune-activation, monocyte-subset frequency, and CD4-memory T-cells in host control of HCV and IFNα- treatment-induced HCV-clearance during HCV-HIV co-infection. We found that CD4 effector memory T-cells positively associated with therapy-induced HCV-decline and anti-viral function, while sCD14 and CD14bright16- monocytes negatively associated with

CD4 effector memory T-cell frequency, HCV-decline and anti-viral function. The data support a role for CD4-memory T-cells in HCV-containment, and connect immune- activation and CD14bright16- monocyte frequency to impaired HCV-clearance.

Chapter 1:

Introduction

Hepatitis C Virus (HCV) and Human Immunodeficiency Virus (HIV)

Clinical impact of HCV and HIV HCV initially entered the human viral arena in the mid-1970s, when it became apparent that there was a distinct virus from hepatitis A and B that was related with chronic infection and cirrhosis, fittingly termed ‘non-A, non-B (NANB) hepatitis’ (2). Research began on this ‘mystery’ virus, resulting in the construct of a cDNA library from samples of a chimpanzee in 1989 (3). The cDNA was observed to hybridize with RNA, not DNA, from the liver of an infected chimpanzee, indicating that this was in fact an RNA virus

(3). Before HCV was identified, AIDS was first termed in 1981, as a sizable fraction of homosexual men were falling prey to opportunistic infections and rare diseases (4, 5). A retrovirus, HIV as we now know it, was determined as the causative agent of AIDS (6-8).

Both HCV and HIV infection are found throughout the world. HCV infection exists in approximately 170 million individuals worldwide, with 60 percent of cases leading to chronic infection (9-11). HIV has infected at least 60 million people worldwide, with 25 million known deaths (12). HIV/AIDS is primarily transmitted via sexual contact (12), but both HIV and HCV can be transmitted via blood to blood contact (13), with HCV infection predominantly resulting from intravenous drug use (14). HCV was also transmitted unknowingly prior to 1990 by blood transfusions, when HCV screening was not performed (14). Lesser common modes of HCV transmission include high-risk sexual activity between men that have sex with men, and vertical transmission that is enhanced if co-infected with HIV (15).

Given the similarity in modes of transmission, there is a significant proportion of HIV infected subjects co-infected with HCV, approximately 9% (16). HIV infection results in a severe immunodeficiency that may lead to lack of control of HIV itself, as well as impaired control of opportunistic infections. CD4 T cell loss is a predictor of disease progression and determinate of immune deficiency (17). Although HIV associated morbidity and mortality has been reduced due to use of antiretroviral therapy (ART),

HCV infection has become a cause of increased morbidity and mortality in HCV-HIV co- infected individuals (18, 19). Furthermore, HIV infection alters HCV disease pathogenesis, accelerating progression to cirrhosis and liver failure (20, 21).

HCV-associated liver disease is a major contributor of liver failure, liver transplantation and hepatocellular carcinoma (HCC) (22). HCC is currently the fifth most common cancer throughout the world, and is one of the leading causes of death, making up 13% of all deaths worldwide (23). In the United States, the rate of HCC cases is increasing swiftly, predominantly due to chronic HCV infection (23). Liver fibrosis, a common occurrence after decades of HCV infection, is also strongly associated with HCC, with

90% of HCC cases presenting in cirrhotic livers (23).

Although HCV therapy advancements have been made, the cost of therapy is high and remains out of reach for many individuals (24). Treatment resistant populations are anticipated, and therapy itself is not anticipated to remove HCV from the population as a

16 whole (25, 26). For these reasons, a better understanding of immune responses to HCV is sought to develop successful vaccine strategies.

HIV virology Overview

HIV structure

Like other retroviruses, the lifecycle of HIV is dictated by the structure of both its RNA and DNA components. The HIV RNA genome is single-stranded and made of three coding regions: gag, pol and env. The four gag proteins make up the capsid, a relatively conserved region of retroviruses, while the pol region is essential for formation of the provirus, making up the viral proteins that are necessary for DNA synthesis and integration (27). Finally, the env region encodes the glycoproteins that make up the HIV envelope: the surface protein gp120 and transmembrane protein gp41 (27). Many viral proteins are necessary to carry out replication, which is enclosed by the HIV capsid. A lipid bilayer envelopes the HIV capsid, which forms when the maturing virus buds from an infected cell.

HIV lifecycle

While gp120 directly binds to the viral receptors CCR5 or CXCR4 on CD4 T-cells (5), gp41 promotes fusion and further entry (27). Inside the cytoplasm of the host cell, the capsid is disassembled partially and the HIV DNA is made from the template RNA genome. Upon completion of reverse transcription, the HIV DNA is sent to the nucleus

17 where it is integrated into the DNA of the host cell, becoming the provirus. The provirus is then capable of transcribing further viral RNA, in a process similar to host cell transcription of mRNA (27).

HIV does not effectively replicate within resting CD4 T-cells, which are found throughout the peripheral blood; therefore, the main site of HIV replication occurs in the lymph node, where it can also infect differentiated macrophages.

HIV clinical manifestations and symptoms

In acute infection, after the first few weeks, HIV titers are high in the blood, and patients experience flu-like symptoms. In this period, a strong cellular and humoral immune response is mounted, resulting in HIV-specific antibodies and cytolytic T-cells that can remain for years. While a sharp decrease in CD4 T-cells is initially seen in acute infection, persistent HIV infection results in a downward slope of CD4 T-cell level, with a subsequent rise in CD8 T-cells within the blood, as seen in Figure 1.1. Chronic infection can either be asymptomatic, or result in lymphadenopathy. When CD4 T-cell levels drops low enough (varying levels below 300cells/mm3) that they are unable to provide their essential function within the immune system, opportunistic infections mount, characterizing progression to AIDS.

Figure 1.1. Clinical Course of HIV Infection. King, Annals of Emergency Medicine, 1994.

HIV treatment: Antiretroviral therapy

The advent of antiretroviral therapy (ART) began with the nucleoside analog AZT, which inhibits reverse transcriptase (28). Since then, as many as 25 drugs have come to market to treat HIV, which can be grouped into six classes: non-nucleoside reverse transcriptase inhibitors (NNRTIs), nucleoside reverse transcriptase inhibitors (NRTIs), protease inhibitors (PIs), fusion inhibitors, CCR5 antagonists (CCR5s), and integrase strand transfer inhibitors (INSTIs) (29, 30). Upon initiation of ART, HIV viral load decreases to undetectable levels within three to six months (29, 30).

HCV virology overview

HCV is a single stranded (ss) RNA (31) enveloped virus of ~60 nm in diameter (32) with

7 distinct genotypes known, identified by the sequence length of the open reading frame

(33). HCV, the only member of the Hepacivirus genus, is of the Flaviviridae family, which includes West Nile, yellow fever and dengue; however, HCV distinguishes itself by being the only pathogen of this family to exclusively infect humans and chimpanzees

(31). While genotype-1 is the most common in North America, Latin America and

Europe, constituting 62-71% of all HCV cases, genotype-2 is also fairly prevalent in these areas (34). However, throughout North Africa and the Middle East, genotype-4 is the most prevalent, and genotype-3 makes up the greatest frequency of HCV cases in

Asia (34).

HCV structure

The structural proteins that make up HCV include the capsid protein C (core), and two envelope proteins, E1 and E2, respectively, and together these structural proteins make up the viral particle (31). While the core compacts the viral RNA, E1 and E2 play vital roles in viral entry and membrane fusion (31). HCV non-structural proteins include the p7 ion channel, the NS2–3 protease, the NS3 serine protease and RNA helicase, the

NS4A polypeptide, the NS4B and NS5A proteins, and the NS5B RNA-dependent RNA polymerase (31). After viral entry into the cell, the non-structural proteins are responsible for viral replication and assembly (31). Of note, both structural and non-structural HCV proteins are either anchored or associated with cell membranes, which indicates that the entire viral life cycle interacts with membranes (35, 36).

HCV attachment and entry

Given that HCV infects hepatocytes (31), this led to investigation of the mechanisms of

HCV liver-tropism, as well as which liver-specific receptors where involved in HCV infection. HCV is regarded as a lipoviral particle, based on its high degree of association with lipoproteins. HCV, upon initial contamination with its host, can be distinguished into density fractions: very-low-density lipoproteins (VLDL), low-density lipoproteins

(LDL) and high-density lipoproteins (HDL) (31). VLDL and LDL have high associations with triglycerides, as well as express apolipoprotein B and E (apoB and apoE, respectively), which provide stability to lipoproteins and allow the absorption of cholesterol (37), and these low-density fractions are also associated with the highest degree cell-culture produced HCV (HCVcc) (38).

HCV entry into the liver occurs via sinusoid capillaries, creating an endothelium made up of both endothelial cells and macrophages. HCV exits the fenestrae of the capillaries and then interacts with hepatic stellate cells (HSCs), presumably via CD81 at the surface of the HSCs, in turn activating production of matrix metalloproteinase MMP-2 (31). MMP-

2 is involved in extracellular matrix degradation, and this could potentially explain, at least in part, for the HCV-associated liver damage (31).

Studies have led to focus on liver host cell molecules that are vital for HCV entry: the tetraspanin CD81, the scavenger receptor class B member I (SR-B1), and the tight

21 junction protein Claudin 1 (CLDN1), as shown in Figure 1.2 (39). CD81 and CLDN1 are known to be expressed within most tissues, including the liver (40-42), while SR-B1 is also expressed within macrophages (43). While HCV infection exclusively occurs within hepatocytes, HCV RNA has also been detected within specific immune cells: B-cells, T- cells, monocytes and dendritic cells, with no replication observed in these cells (31).

Furthermore, HCV can directly interact with immune cells that express the aforementioned molecules that are vital for HCV entry. Natural killer (NK) cells express

CD81 and have been shown to interact with HCV virions via E2 (44); however, the role of HCV on NK cell effector function is not completely understood.

It has also been shown that the first extracellular loop of CLDN1 can interact with HCV

(45). In fact, HCV-infected liver tissue express elevated levels of CLDN1 compared to normal liver tissue, and in vitro studies have demonstrated that HCV modulates CLDN1 in Huh7.5 hepatoma cell lines (39). HCV glycoproteins, including E2, have been shown to interact with CD81 and SR-B1, and these interactions allow HCV entry into hepatocytes (31, 46, 47). SR-B1 can also recognize and bind to HDL of the surface of

HCV, promoting HCV infectivity (48). Once bound to CD81, the tight junction CLDN1 is formed, and HCV enters the hepatocyte via clathrin-dependent endocytosis (42, 49).

However, the mechanisms of these specific liver host cell interactions with HCV have not been fully elucidated.

HCV replication, assembly and release

As previously mentioned, the non-structural proteins that make up HCV are responsible for the replication and assembly phases of the life cycle. The 5′ non-coding region

(NCR), which is highly conserved among HCV isolates(50), has been shown to be required in its entirety for RNA amplification (1), containing a ribosome entry site viral for translation initiation (51). However the 3′ NCR appears to possess a nonessential variable region (1). p70, serine protease responsible for cleaving and processing the

HCV-encoded polyprotein NS5B is absolutely required for HCV RNA replication, act as the RNA polymerase (1). NS5B uses the positive-strand of the HCV genome as a template to make a negative-strand intermediate, which in turn acts as a template to create many new individual genomes (1). NS4B and NS5A together contribute to induction of membranous web formation, which has been demonstrated to act as a scaffold for HCV replication complex assembly (1).

The long-awaited advent of cell-culture produced HCV (HCVcc) limited the understanding not only of the mechanisms of the HCV life cycle, but also of the HCV immune response. Finally, Japanese-fulminate hepatitis-1 was discovered, and was infectious to the human hepatoma cell line Huh-7 (38), and was capable of producing

HCVcc. A single point mutation in the dsRNA sensor retinoic acid-inducible gene-I

(RIG-I) of Huh-7 cells, creating Huh-7.5 cells, has been shown to increase the permissiveness to HCV infection (1), due to muting the innate IFN-response to HCV

(52). Use of this HCV-permissive Huh-7.5 cell line has allowed creation of in vitro assays to better understand the immune response to HCV.

Figure 1.2. HCV Lifecycle. Brett D. Lindenbach and Charles M. Rice. Nature. 2005. (1)

HCV clinical symptoms and manifestations

Many who are initially infected with HCV are unaware, because the virus does not typically present symptoms until after 3 to 12 weeks of exposure, if at all (53). However, in instances in which symptoms occur, there is general malaise, loss of appetite and jaundice (53). Approximately 1 to 2 weeks after HCV exposure, RNA levels are detectable, followed by increasing levels of markers of liver inflammation at 8-12 weeks

(11, 22). These markers include liver enzymes aspartate amino transferase (AST), as well as alanine amino transferase (ALT). While spontaneous clearance of HCV occurs in 20-

25% of cases, most initial infections result in persistent, or chronic viral infection (9).

Chronic HCV infection, defined by at least 6 months of infection (22), can result in liver damage and fibrosis, as marked by heightening levels of serum AST and ALT, and indirectly by lower levels of circulating platelets and serum albumin. In turn, chronic infection and resultant liver damage is a major driver of hepatocellular carcinoma and liver transplant (54).

HCV treatment HCV therapies have included PEGylated (PEG) interferon (IFN) in combination with ribavirin (RBV) (55). PEG makes IFN last longer in the body (55). RBV is a guanosine analog used to stop viral RNA synthesis (56, 57). While studies have shown that there are varying patterns of moderate, minimal or no effect of RBV as a monotherapy for HCV

(56, 58), the addition of RBV to IFN-therapy improves outcome (59). Efficacy of PEG-

IFN, in combination with RBV has led to sustained control of HCV in approximately

50% of patients (59, 60). Of note, the sustain control of HCV with these therapies was significantly lower for those co-infected with HIV (61). These IFN-based therapies were associated with many side-effects, with lower treatment responses in individuals with advanced liver disease (62).

Direct Acting Antiviral (DAA) HCV therapy

Major advancements in HCV therapy have occurred, resulting in highly effective IFN- free direct-acting antiviral (DAA) agents. The first DAA therapies included protease inhibitors against NS3/4a, serine protease responsible for cleaving and processing the

HCV-encoded polyprotein (63). Protease inhibitors boceprevir, which binds to the NS3 active site, and telaprevir, which inhibits NS3/4A activity, were both used in combination with IFN/ribavirin (RBV) for 24-48 weeks (64), and were most effective against HCV-1

(63). These therapies led to 50-70% SVR (64). The newer protease inhibitor, semeprivir, inhibits NS3/4A activity like telaprevir before it, but with enhanced binding affinity and specificity (63).

More recently, the RNA polymerase inhibitor Sofusbuvir, a nucleotide analogue, and

Ledipasvir, an NS5A inhibitor, have been shown to be effective against HCV-1-4 (63).

Sofusbuvir-Ledipasvir have shown effectiveness as well in HCV-HIV co-infected subjects (65). Sofusbuvir can be used with or without IFN (66). Sofusbuvir and

Ledipasvir, with or without RBV for 12-24 weeks, with some regimens as short as 8 weeks(67), have resulted in 94-99% viral clearance (66). However, the cost of this therapy is approximately $84,000, which prevents its access to many, and treatment- resistant populations still remain (67).

The Immune Response to HCV

Overview of the interplay of the innate and adaptive immune systems

The potential first response to HCV may be by hepatocytes via dual roles of TLR3, which recognizes dsRNA within endosomes, and RIG-1, which identifies a region of the HCV

3’NCR. The engagement of TLR3 and RIG-1 leads to recruitment of Toll-IL-1-receptor domain-containing adaptor inducing IFN-β (TRIF), ultimately leading to production of

IFN-β, a type-I interferon (11). Plasmacytoid dendritic cells (pDCs) are also capable of release type-I interferons, acting as the predominant producer of IFNα. In turn, IFN-β creates an anti-viral state that directly and indirectly affects nearby cells by interaction with the IFNα/β receptor. This interaction leads to induction of interferon-stimulated genes (ISGs) via the JAK/STAT pathway (11).

Conventional dendritic cells (cDCs) are located within the tissue and emigrate to lymph nodes where they interact with and prime T-cells to a Th1 response via production of polarizing cytokines, including IL-12 (11). Natural killer (NK) cells are also key players in the innate response to HCV. Many studies have demonstrated that genetic factors are associated with HCV-directed NK cell IFNγ release and cytotoxic function, based on expression of killer cell immunoglobulin-like receptor (KIR) (11, 68, 69).

Upon activation during the acute response to HCV infection, within 5-9 weeks, HCV- specific T-cells and antibodies are generated (70, 71). While successful HCV clearance can be achieved without a humoral response (72), HCV-specific T-cells are vital (70, 73) via IFNγ production and cytotoxicity (74, 75). Notably, IFNγ appears to act as the more effective anti-viral mechanism to clear HCV (75). CD4 T-cells specifically play a strong role in this ‘acute’ response (characterized clinical time window of 6 months), producing

IL-2 and IFNγ (76, 77), and subjects who have little to no CD4 T-cell response develop chronic HCV infection (78, 79). Memory CD8 T-cells are detectable upon induction of

HCV-specific CD4 T-cell responses, followed by subsequent HCV decline (70, 73, 77).

Without CD4 T-cells, the protective anti-viral activity of CD8 T-cells would not occur, supported by in vivo depletion of CD4 T-cells from HCV-infected chimpanzees (80).

HCV works to inhibit this anti-viral response via many mechanisms. NS3/4A, cleaving

TRIF, while HCV core inhibits JAK/STAT signaling and binds to DCs to prevent IL-12 production (11). Both HCV core and NS3 also activate monocytes via TLR2, leading to induction of TNFα; TNFα then inhibits IFNα production, as well as promoting pDC

28 apoptosis, blocking the main source of IFNα (11). Additionally, HCV NS5A leads to induction of IL-8, a pro-inflammatory cytokine that inhibits ISG expression (11). HCV

E2 also binds to NK cells via interaction with CD81, inhibiting NK cell activity (44, 81).

In chronic HCV infection, HCV-specific CD4 and CD8 T-cell functions are impaired (70,

73, 77). These HCV-specific T-cells with impaired anti-viral function are characterized by PD-1 expression, an inhibitory receptor (82, 83). PD-1 on T-cells interacts with the

PD-1 ligand, PDL-1, found on sinusoidal endothelial cells, Kupffer cells, stellate cells, and type-I-exposed hepatocytes, leading to not only impaired responses, but also T-cell apoptosis (84). IL-10 production, by T-cells, NK cells, and TLR-2-activated monocytes

(85, 86), has been shown to be elevated within chronic HCV infection (85). IL-10- producing CD8 T-cells have been found in the liver and have been shown to mute IFNγ production, as well as the proliferation of HCV-specific CD4 T-cells (87). IL-10 can also inhibit IFNα production, mediate pDC apoptosis (86, 88), ultimately muting the overall anti-viral immune response. Further understanding how HCV works to inhibit immune function will allow development of better anti-viral strategies for therapy and vaccines.

Natural killer (NK) cells and the potential role for IL-7 on anti-viral effector function

NK cell activating and inhibitory receptors

NK cells have been well described to be regulated by the balance of activating and inhibitory receptors. In normal healthy conditions, NK cells are tightly controlled by inhibitory signals that override activating receptors that prevent NK cell activation effector function against host cells (89). These inhibitory NK receptors (NKRs) include killer immunoglobulin-like receptors (KIRs) and the C-type lectin of the CD94/NKG2 family (NKG2A) (89). The predominantly inhibitory KIR family is highly diverse, binding to MHC I ligands that are also highly diverse and polymorphic, together providing diversity in immune responses (89).

NK cell KIR expression and recognition of self MHC 1 ligands constitute the ‘missing- self’ hypothesis that aims to explain NK cell activation. When target cells, such as virally-infected or tumor cells, downregulate MHC 1 ligand expression, NK cells are poised to become activated (90). The activating NKRs typically require a second signal to exert effect, such as loss of inhibition or inflammatory cytokine stimulus (89, 90).

Included in the activating NK receptor repertoire are the natural cytotoxicity receptors

(NCRs) NKp30, NKp44 and NKp46, as well as NKG2C/D and certain KIRs (91). There are additional surface receptors that can also regulate NK cells that are not exclusively expressed on NK cells.

Toll-like receptors (TLRs) are pathogen-recognition receptors, triggered upon exposure to pathogen-associated molecular patterns. TLRs, which have well-documented expression on DCs, monocytes and macrophages (92), have also been demonstrated to be expressed on NK cells, specifically TLR1, 2, 3, 7 and 9 (93-96). This provides NK cells with additional means of activation independent of target cells (97). TLR7 provides the cell ability to recognize HIV (90), and may also offer detection of HCV (98). TLR2 offers detection of HIV replication (90) and engagement with HCV (99). TLR3 has also been shown to provide antiviral responses to HCV in vitro (99). Upon exposure to TLR ligation, both DCs and NK cells are activated and engage the other in bi-directional crosstalk which helps modulate the anti-viral immune response. TLR stimulation leads to induction of type-I interferons (IFNs) and other inflammatory cytokines (100, 101). NK cells can also express tumor necrosis factor (TNF)-related apoptosis-inducing ligand

(TRAIL) and Fas ligand (FasL/CD95), which are responsible for extrinsic induction of cell death (89, 102, 103). In summary, NK cell activation and effector function are controlled by downregulation of inhibitory receptors, upregulation of activating receptors and stimulus from inflammatory cytokines, including IL-12, IL-15 and IFNs that are induced by viral infection (89).

NK cell response to chronic viral infection

Natural killer (NK) cells can contribute to viral control via cytokine secretion, predominantly IFNγ and TNFα, and cytolytic mediators released from intracellular granules (104). These anti-viral NK effector functions are regulated by activating and inhibitory receptors on NK cells (105). NK cells can be found at highest levels in the

31 blood, liver and spleen, and also in lesser quantities in the lymph nodes and other inflamed sites (104). NK cells have been traditionally divided into subsets based on expression of CD56 (NCAM-1) and CD16 (Fc Receptor): CD56-16+ (CD56neg),

CD56dim16+ (CD56dim) and CD56bright16dim/- (CD56bright) NK cells (106). While the

CD56dim subset is the most cytotoxic and most predominant in the blood (107), accounting for nearly 90 percent of NK cells in the blood, the CD56neg and CD56bright subsets can also carry out cytotoxic function (108-110).

Although about 5-10 percent of NK cells in the blood are CD56bright (107), this subset is found to be more predominant in secondary lymphoid tissue, with greater expression of secondary lymphoid homing receptors than the other NK subsets (111). The CD56bright

NK subset has been described as immunoregulatory, as it is the predominant cytokine- secreting NK subset, and also interacts with dendritic cells (DCs) in secondary lymphoid sites (111-114). The effects of NK cytokine secretion determine the adaptive immune response. Increased NK pro-inflammatory cytokine production, such as TNFα and IFNγ, leads to a stronger Th1 response (112, 115). Likewise, reduction of these NK proinflammatory cytokines or increased production of immunosuppressive cytokines by NK cells, such as IL-10, can dampen the adaptive immune response to viral infection (89,

116). Because of the multifaceted roles of NK cells, from immunoregulation to lysing of virus infected cells, a better understanding of how they become activated and/or polarized may lead to key antiviral strategies.

NK cell function has been shown to be important in control of HCV and HIV infections

(106, 116, 117). Studies have shown that NK human leukocyte antigen (HLA) and KIR repertoire has been associated with HCV infection outcome, and also HIV progression, conferring protection with specific polymorphisms disposing to enhanced effector function (68, 104, 118). Within HIV infection, the expression of KIR3DS1, an activating receptor, and KIR3DL1, an inhibitory receptor, on NK cells, together with their respective putative ligands, have been associated with slower progression of HIV disease

(119). Furthermore, in vitro studies have demonstrated that individuals with high NK

KIR3DS1 expression have heightened IFNγ production, cytotoxicity and ability to inhibit viral replication (120).

Multiple groups have shown altered NK cell subset frequency and phenotype during chronic HCV, HIV and HCV-HIV infections, with increased frequency of CD56bright NK cells and expansion of the lesser known, exhausted CD56neg NK cell population (92, 121,

122). These perturbations in NK cell subset frequency are also accompanied by alterations in NK cell effector function (105, 121). We currently know that strong inducers of NK cell IFNγ production and secretion include IL-12, IL-15 and IL-18, with

IL-15 also critically vital in NK cell development and maturation (44, 81, 123). Within

HIV infected subjects, there is reduced NK IFNγ production in response to in vitro stimulation with IL-12 and IL-18 (121).

Similar changes in NK cell function have been seen in HCV infection. Reports have shown that NK cell IFNγ secretion is reduced in chronic HCV infection, and this is

33 associated with inability to clear the virus (89, 92). HCV has been demonstrated to inhibit NK cell cytotoxic function and IL-2-induced IFNγ production via CD81 on NK cells binding to HCV E2 (44). Additionally, NK cells within chronic HCV infection are described as polarized toward cytotoxicity, correlating with alanine aminotransferase

(ALT) and aspartate aminotransferase (AST) levels (92, 122). Furthermore, this cytolytic polarization is also accompanied with decreased selectivity for infected target cells (122).

It therefore may be desirable to find targets that improve the selectivity of NK cytolytic activity and skew this polarization to increased IFNγ production that may in turn provide for better control viral infection (Figure 1.3).

The role of IL-7 and its receptor on NK cell anti-viral effector function

Given the indispensable role of IL-7 in T-cell development (124), much work has focused on the use of IL-7 within HIV and HCV infections. IL-7 is a member of the IL-2 γ-chain cytokines, produced by stromal cells in the bone marrow, thymus and lymph node, signaling through the JAK-STAT5 pathway (125, 126). IL-7 has the ability to co- stimulate T-cell activation, via enhancement of proliferation and cytokine production

(124). Importantly in the case of viral infection, IL-7 leads to a strong type 1 immune response by its upregulation of IFNγ and IL-2 production (127, 128). Work has also shown that IL-7 is able to synergize with IL-12 to induce T-cell proliferation and IFNγ production (129).

Within HIV infection, we see CD4+ T cell lymphopenia, chronic activation of the immune system, and the impairment of T cell production (130). Vital in T-cell

34 homeostasis, IL-7 levels have been shown to rise in CD4 T-cell lymphopenic conditions, including HIV infection (131-133). Upon antiretroviral therapy (ART) institution, as

CD4 T-cell counts rise, the respective IL-7 serum levels lower to normal levels (126, 130,

131). Work has shown that this reduction of IL-7 to normal levels while on ART is achieved by increases in the IL-7 receptor (CD127) expressed on T-cells, to carry out receptor-mediated clearance of IL-7 (126). Meanwhile, CD8+ T-cells that lack CD127 are associated with disease progression and immune activation (130).

Within HCV infection, CD8 T-cell CD127 expression has been negatively associated with HCV viral load and alanine aminotransferase (ALT), a marker of liver inflammation

(134, 135). In vitro HCV-specific CD8 T-cell CD127 expression is positively associated with production of IFNγ in samples from HCV infected subjects (134, 135). After in vitro challenge with HCV peptide, CD8 T-cell CD127 expression also negatively correlated with pro-apoptotic marker Bim and positively correlated with pro-survival marker Mcl-2

(135). Similarly, T-cell CD127 expression also positively correlates with pro-survival marker Bcl-2 (134). These results suggest that CD127 expression on CD8 cells confers protection and improved function during HCV infection.

Since NK and T cells have overlapping functions, including cytolytic activity and proinflammatory cytokine production, it is possible that IL-7 may have similar effects on NK cells. In fact, CD127 is expressed on NK cells, with most significant levels found on the

CD56bright NK subset (136). NK cells treated with IL-7 lead to induction of lymphokine- activated killer (LAK) activity, proliferation, and TNFR expression (137). Similarly as

35 seen with IL-7 treatment of T cells, BCL-2 is upregulated within CD56bright NK cells cultured with IL-7 (136). In HIV infection with suppressed viremia, it has been shown that upon treatment of NK cells from with IL-7 there is increased cytolytic activity (102).

However, the effect of IL-7/CD127 on NK cells within HCV infection is unknown. We evaluated here whether CD127/IL-7 signaling skews NK polarization to more IFNγ production and results in better control of HCV (Figure 1.3).

NK cell interactions with dendritic cells and monocytes

NK cells aide in dendritic cell and monocyte maturation

NK cells co-exist and interact in reciprocal relationships with other innate immune cell types throughout circulation and secondary lymphoid tissue. Notably, NK cells exchange in bi-directional crosstalk with dendritic cells and monocytes. NK cells, upon stimulation with cytokines or recognition of ligands for respective activating receptors (93), provide iDCs with signals to mature (93, 114, 138-141). CD56bright NK cells impact DC maturation, lysing immature DCs (iDCs) and promoting DCs to become strong professional antigen processing cells, effective priming of T cells which is key to the formation of a strong adaptive immune response (106, 138). These NK cells, at sites of inflammation, select suboptimal, low MHC class 1-expressing, iDCs to be targeted for cytolysis via the natural cytotoxicity receptor (NCR) NKp30 (138, 139). As aforementioned, the predominant NK cell subset responsible for this role in accessory cell maturation are CD56bright NK cells, expressing high levels of NKG2A and little to no

KIRs (138, 139). Ultimately, this quality control process results in mature DCs,

36 enhancing the ability of DCs to activate a T-cell (114). CD56bright NK cells can also affect the maturation and effector functions of other immune cells, including macrophages, granulocytes and other lymphocytes key in fighting infection (104). Circulating monocytes are bone marrow-derived phagocytic cells that play a key role within the innate immune response to bacterial, fungal, parasitic and viral infections (142-146), that ultimately give rise to macrophages in specific tissue sites.

DC-dependent NK cell effector function

While NK cells play a key role in DC maturation, DCs are also vital in shaping an effective NK cell response. In turn, mature DCs activate these NK cells to carry out their effector functions, to directly kill HCV-infected cells and secrete TNFα and IFNγ, contributing to an antiviral immune response (115). These stimulations can further prompt NK cells to carry on the DC editing process. Prominent inflammatory cytokines released by DCs to stimulate NK cells include IL-12 and IL-15 (97). In fact, IL-12 secretion from DCs is crucial for NK cell induction of IFNγ release and improving NK cell cytolytic activity (97).

Monocyte-dependent NK cell effector function

Upon stimulation, monocytes and macrophages are also capable of regulating NK cell function via secretion of cytokines and direct cell contact. Monocytes have also been demonstrated to be critical in regulating NK cell effector functions. Via inflammasome- dependent secretion of IL-18 upon interaction with HCV-infected hepatoma cells, monocytes activate NK cells to produce IFNγ, which in turn inhibits HCV replication

(117). In fact, when CD14+ cells were depleted from PBMC cultures with or without

HCV infection, the level of intracellular IFNγ within CD56bright NK cells was significantly reduced compared to cultures with HCV infection (117), indicating a critical role for monocytes in HCV-directed NK cell effector function. Together, the mounting data suggest a need to better understand the role of monocyte and NK cell interactions to initiate and maintain an effective anti-viral immune response.

The role of immune activation on the immune response to chronic viral infection

Soluble immune activation mediators and monocytes in HCV and HIV

It has been well demonstrated that chronic HCV and HIV infections each lead to a state of immune activation, leading to higher levels of pro-inflammatory cytokines and soluble factors, such as sCD14 and IL-6 (147-152). HIV infection also increases the state of immune activation (16, 19). Immune activation and inflammation are known predictors of HIV-1 disease progression and mortality (153-155). Similarly, soluble mediators of immune activation have been associated with poor outcome in HCV infection (148). We and others have shown that sCD14 negatively predicts outcome to HCV therapy (148,

151). However, the effect of persistent immune activation on the innate immune system is poorly understood (156).

Monocytes are characterized based on expression of CD14, the lypopolysacchirade (LPS) receptor (146), and have been further dissected into three subsets based on surface expression of CD16: CD14bright16- classic (the predominant monocyte subset),

CD14bright16+ intermediate and CD14dim16+ patrolling monocytes (146, 157). Circulating monocytes and tissue macrophages have been shown to be associated with initiation and progression of HIV-associated morbidities (158-161) as well as poor outcome to HCV therapy (149). Within viremic HIV-1 mono-infection and HCV mono-infection, the proportions of intermediate and patrolling monocyte subsets are increased (157, 162).

There is a report of normalization of the monocyte subset distribution upon HAART in

HIV-infected individuals (163). Consistent with the observed increase proportion in

CD14bright16+ and CD14dim16+ monocyte subsets within HIV infection and their roles in viral infections, culture of PBMC-derived monocytes with the HCV replicon system led to an increased shift in both these monocyte populations (117).

Each subset is functionally distinct from the other due to differential expression of homing markers, antigen presentation abilities and production of cytokines upon bacterial

TLR stimulation (146, 164). Classic monocytes produce high levels of IL-6, IL-8, CCL2 and CCL3, moderate amounts of IL-10 and minimal TNFα, as shown in vitro, while

CD14brightCD16+ monocytes produce inflammatory cytokines such as TNFα and IL-1β in addition to moderate amounts of IL-10, upon stimulation with LPS (146, 164). These inflammatory CD14brightCD16+ monocytes have also been associated with disease progression in viremic HIV-1-infected subjects (163).

Patrolling monocytes have been reported to express high surface expression of TLR7 and

TLR8 that are capable of sensing nucleic acids and viruses (146). While response to bacterial signals such as LPS and TLR2 stimulation led to weak patrolling monocyte

39 response, presence of measles and HSV-1 resulted in high amounts of proinflammatory cytokines TNFα, IL-1β and CCL3 (146). These cells are able to explore the vasculature, attaching to the endothelium and carrying out a crawling-like movement, unlike

CD14bright monocytes (146). These human CD14dim patrolling monocytes are homologous to the Gr1- patrolling monocytes, which conserve expression of integrins, including lymphocyte function-associated antigen-1 (LFA-1) (146, 165).

The effect of immune activation on NK cells

The role of immune activation on NK cell function has not been clear, and it is unknown how immune activation affects NK cell CD127-mediated activity and control of viral infection. However, some studies have demonstrated a role of IL-6 and sCD14 on NK cell phenotype and function. While we see heightened levels of sCD14 in HCV infected subjects in this data set (149), and also in other previous studies (148), the biological significance of increased sCD14 on NK cell phenotype and function is not understood.

However, it has been established that both elevated sCD14 levels and NK cell activation at the onset of HCV therapy both associated with poor outcome in HCV infection (148).

Similarly, sCD14 levels were also positively associated with NK cell activation (CD38 expression), which correlated with poor prognosis in HIV subjects (150). Furthermore, sCD14 levels also correlated with higher frequency of the dysregulated CD56neg NK cell subset (148).

IL-6, which is heightened in both HIV and HCV infections (147), leads to modest proliferation of NK cells, as well as increased CD69 expression and cytolytic activity

(166). However, IL-6 is unable to induce NK cell IFNγ production (166). Therefore, in the proposed model (Figure 1.3), IL-6 and sCD14, immune activation markers/mediators, are proposed to further polarize NK cells of HCV subjects toward increased cytotoxic function and reduced IFNγ production. Here, we will focus on the novel findings of the role CD127 and IL-7 on NK effector function within HIV, HCV, and HCV-HIV infections, and offer guidance for future studies on the mediators of immune activation and their potential effects on NK cell effector function in chronic viral infections (Figure 1.3).

Figure 1.3 Model. In chronic HCV, NK cells are polarized to cytotoxicity, which leads to liver inflammation and inability to clear virus. There is increased loss of viral control and inflammation in the setting of immune activation, which is induced in HIV and HCV infections. HCV and HIV can activate monocytes including interaction with TLRs, which can lead to production of sCD14 and IL-6, mediators of immune activation. While NK cells contribute to monocyte maturation, monocytes also produce IL-10 which can suppress NK cell anti-viral function.CD127/IL-7 will lead to increased NK IFNγ and better viral control.

Chapter 2:

CD56bright NK cell IL-7Rα expression negatively associates with HCV level while IL-

7-mediated NK cell cycling and cytolytic function are impaired during HCV and

HIV infections

*This article was originally submitted to Journal of Immunology. Chelsey J. Judge, Lenche Kostadinova, Kenneth E. Sherman, Adeel A. Butt, Yngve Falck-Ytter, Nicholas T. Funderburg, Alan L. Landay, Michael M. Lederman, Scott F. Sieg, Johan K. Sandberg and Donald D. Anthony (2016). CD56bright NK cell IL-7Rα expression negatively associates with HCV level while IL-7-mediated NK cell cycling and cytolytic function are impaired during HCV and HIV infections. Submitted.

Abstract

Several lines of evidence support the concept that natural killer (NK) cells play an important role in control of HCV infection via cytokine secretion and cytotoxicity. IL-7 is a homeostatic cytokine with a role in T-cell development, activation, proliferation and cytokine secretion. The IL-7 receptor α chain (CD127) is expressed on NK cells, with greatest abundance on the CD56bright subset. Here, we initially measured CD127 expression on CD56bright, CD56dim or CD56neg NK cell subsets of 25 uninfected donors,

34 chronic HCV-infected treatment-naïve, 25 HIV-infected, virally suppressed on antiretroviral therapy (ART) and 42 HCV-HIV co-infected subjects on ART.

Interestingly, CD127 expression on CD56bright NK cells negatively correlated with HCV plasma levels in HCV mono-infection and HCV-HIV co-infection. IL-7 induced CD69 expression, enhanced IFNα-induced CD69 expression on CD56bright NK cells, and this was impaired in HIV-infection. IL-7 induced NK cell IFNγ in samples from all groups similarly. IL-7 induced Bcl-2 expression and cell cycling of CD56bright NK cells, and this effect was impaired in HCV and HIV infected subjects. While IL-7 induced CD56bright

NK cell degranulation appeared intact within all groups, we observed impaired IL-7- activated NK cell cytolytic function in HCV and HIV infected subjects. Finally, IL-7- induced pSTAT5 signaling was impaired in NK cells of subjects with chronic viral- infection, which was reversible upon 6 months of viral-suppression with IFN-free HCV- therapy. These results implicate IL-7-dependent NK cell activation and effector function as contributing to control of chronic HCV-infection.

Introduction

HCV infection is present in approximately 170 million individuals worldwide, with 60-85 percent of acute infections leading to chronic infection (9-11). HCV therapy has improved significantly in recent years, although the cost of interferon (IFN) free antiviral therapy remains a barrier for many (24). Additionally, therapy itself is not anticipated to remove HCV from the population as a whole (25, 167). For these reasons, a better understanding of the host immune response to HCV is sought to develop successful vaccine strategies.

Given overlapping modes of transmission, there is a significant proportion of HIV infected persons co-infected with HCV (16). Although HIV-associated morbidity and mortality has been reduced due to the use of antiretroviral therapy (ART), HCV infection is a cause of increased morbidity and mortality in HCV-HIV co-infected individuals (18,

19). Furthermore, HIV infection alters HCV disease pathogenesis, accelerating progression to cirrhosis and liver failure (20, 21).

NK cells are important in immune control of HCV and HIV infections (106, 116). HLA and killer immunoglobulin-like receptor (KIR) repertoires have been associated with

HCV infection outcome, and also with rates of HIV disease progression (68, 91, 118).

NK cells are readily found in the blood, liver and spleen, and in lesser quantities in the lymph nodes and inflamed tissues (91). NK cells are divided into three main subsets based on expression of CD56 (NCAM-1) and CD16 (FcγRIIIA): CD56brightCD16dim/-

(CD56bright), CD56dimCD16+ (CD56dim) and CD56neg16+ (CD56neg) NK cells (106, 109).

About 5-10 percent of NK cells in the blood are CD56bright (107), and this subset is more common in secondary lymphoid tissue and the liver (111). The CD56bright subset is

45 considered less mature than the CD56dim and CD56neg subsets, and is less cytotoxic, yet is proficient in cytokine production (112, 115). NK cells can contribute to viral control via cytokine secretion, predominantly IFNγ and TNFα, chemokine secretion, and cytolytic function (91). NK cell IFNγ production is reduced in chronic HCV and HIV infections

(121), and this is associated with inability to clear HCV (89, 92). Because of the multifaceted roles of NK cells, from immunoregulation to cytolysis of virally-infected cells, a better understanding of how they are activated during chronic viral infection is likely to inform antiviral vaccine and treatment strategies.

IL-7 is produced by stromal cells in the bone marrow, thymus, lymph node, and liver.

The IL-7 receptor is composed of the common γ chain (CD132), and the high affinity α receptor (CD127), and signals through the JAK-STAT5 and AKT pathways (125, 126).

Given the indispensable role of IL-7 in T-cell development (124), much work has focused on the role of IL-7 in HIV and HCV infections. Loss of CD127 expression on both CD4 and CD8 T cells has been associated with inability to resolve both acute and chronic

HCV infection (134). Additionally, CD127 expression on HCV-specific CD8 T cells is negatively correlated with HCV viral levels, and positively correlated with intracellular

IFNγ expression (134, 135). Little is known, however, about the role of IL-7 in regulating

NK cell function during chronic HCV or HIV infections. Here, we evaluated IL-7 dependent NK cell effector function in subjects infected with HCV, HIV or co-infected with both HCV and HIV. Together, these data support a role for IL-7-dependent NK effector function in control of chronic viral infection, and highlight yet another potential virally-associated impairment of host immune surveillance.

Materials and Methods

Study subjects

Thirty-four HCV-infected subjects (antibody positive ≥6 months, HCV RNA positive, genotype 1) naive to therapy, 42 HCV+HIV+ co-infected subjects, 25 HIV-infected subjects (HIV ELISA and Western Positive, Prior HIV RNA positive, HIV-1 RNA <50 copies/ml on ART for at least 8 weeks, HCV RNA negative, CD4 cell count >200/mm3), and 25 age-range-matched uninfected donors (UD) were enrolled (Table 2.1). The HCV-

HIV co-infected subjects met above criteria for HCV and HIV infection, and were infected with genotype 1 HCV. Duration of HCV infection was estimated based on first year of known intravenous drug use or blood transfusion before 1992. All subjects gave

Institutional Review Board approved written informed consent at either the Veterans

Affairs Medical Center, University Hospitals of Cleveland, or at one of the AIDS Clinical

Trials Group sites. Aspartate aminotransferase (AST), alanine aminotransferase (ALT), platelet count (PLT), albumin, plasma HCV RNA, and plasma HIV RNA levels were determined at the corresponding clinical laboratory. The aspartate aminotransferase

(AST) to platelet (PLT) ratio index (APRI) was calculated as [(AST/upper limit of normal)/PLT] × 100 (168).

Table 2.1. Clinical Parameters of Study Subjects.

Variable HCV HCV-HIV HIV Controls values

N 34 64 25 25

Age, years 58 (51-65) 47 (24-64) 51 53 (23-81)

Male sex, % 97 81 88 94

Race:

Black, % 47 47 28 56

White, % 50 41 66 32

other, % 3 12 6 12

1,500,019 9,527,998 Plasma HCV (60,156- (5,730- 0.0004 level, IU/ml 4,404,430) 72,600,000) Plasma HIV

RNA level, Undetectable Undetectable copies/ml

Serum albumin 3.9 (2.8-4.5) 4.3 (3-5.1) 4.1 (3.5-4.6) 4.1 (3.5- <0.0001 level, g/dL 4.5)

Platelet count, 223 (56–342) 203 (93-394) 239 (81-418) 227 (152- 0.20 k/cmm3 313)

Serum AST 56.3 (20-243) 60.4 (19-172) 25.25 (11- 26.8 (16- <0.0001 level, UL 70) 47)

Serum ALT 64 (20–303) 75 (19-239) 37 (13-85) 28 (9-56) <0.0001 level, UL

Ex vivo NK cell CD127 expression

Cryopreserved peripheral blood mononuclear cells (PBMC), freshly isolated PBMC or freshly isolated NK cells (negative bead selection method (STEMCELL Technologies; depleting CD3/CD4/CD14/CD19/CD20/CD36/CD66b/CD123/HLA-DR/glycophorin A– expressing cells; median purity >95%), were analyzed by viability LIVE/DEAD-yellow dye (Invitrogen, Grand Island, NY). Lymphocytes were identified by forward and side scatter and NK cell phenotype was assessed using the following mAbs: anti-CD3-CD14-

CD19-AlexaFluor700 (UCHT1, M5E2, Biolegend, San Diego, CA), anti-CD7-PerCP-

Cy5.5 (BD Biosciences, San Jose, CA), anti-CD56-PE-Cy7(B159, Biolegend), anti-

CD16-APC-H7 (3G8, BD Biosciences), and anti-CD127-BrilliantViolet421 (A019D5,

Biolegend), or isotype controls. Flow cytometric data was acquired on a BD LSRII and analyzed with TreeStar FlowJo software.

IL-7 induction of Ki67, BCL2, IFNγ and pSTAT-5

Fresh PBMC were cultured in presence or absence of 10 ng/ml IL-7 or 500 U/ml IFNα overnight. In some assays, IL-7-treated cells were incubated additionally with wortmannin (500nM; PI3K inhibitor; Sigma-Aldrich) or N’-[4-Oxo-4H-chromen-3- yl)methylene] nicotinohydrazide (500μM; P-STAT5 inhibitor; EMD Millipore, Billerica,

MA, USA). Cells were then surface stained, fixed, and permeabilized with a saponin- based buffer (BD Biosciences), followed by incubation with anti-Ki67-PE, anti-Bcl2-

FITC or anti-IFNγ-FITC (BD Biosciences) for 40 minutes on ice. For detection of phospho-epitopes, fixed cells were permeabilized with a methanol-based buffer (BD

Biosciences) and stained with anti-phospho-Stat5 Alexa Fluor 647 (Y694) (BD

Biosciences) and analyzed on a BD LSRII flow cytometer.

In vitro NK cell CD127, CD69, FasL, TRAIL, NKp30, NKG2A and IFNαR1 expression

Purified NK cells were cultured in absence or presence of 10 ng/ml IL-7 (unless otherwise stated), IFNα (500 U/ml), or IL-12 (1 ng/ml) in 96-well round bottom plates for 20 hours. In some assays, IL-7-treated cells were cultured additionally with either wortmannin or P-STAT5 inhibitor as stated above. NK cells were then stained with

LIVE/DEAD-yellow viability dye (Invitrogen), anti-CD3-14-19-AlexaFluour700

(UCHT1, M5E2, Biolegend), anti-CD7-PerCP-Cy5.5 (M-T701, BD Biosciences), anti-

CD56-PE-Cy7 (B159, Biolegend), anti-CD16-FTIC (3G8, BD Biosciences), anti-CD127-

BrilliantViolet421 (A019D5, Biolegend), anti-CD69-APC-Cy7 (FN50, Biolegend), anti-

FasL-BrilliantViolet421 (DX2, Biolegend), anti-TRAIL-PE (RIK-2, BD Biosciences), anti-NKp30-AlexaFluor647 (p30-15, Biolegend), anti-NKG2A-PerCP (IC003C, R&D

Systems, Minneapolis, MN), and anti-IFNαR1-PE (MMHAR-1, Interferon Source), or isotype controls and analyzed on a BD LSRII flow cytometer.

IL-7 and sCD127 ELISA

Serum was evaluated for IL-7 levels by enzyme-linked immunosorbent assay (R&D

Systems).

A 96-well polystyrene plate was coated with 50 μl mouse anti-human CD127 (100 ng/ml,

R&D Systems) in PBS overnight. The next day, the plate was washed (PBS-Tween) and blocked for 2 hours at room temperature with PBS + 1% BSA. The plate was washed and

50 μl serum or standards were incubated for 3 hours at room temperature. After washing, the plate was incubated with 5 μg/ml biotin poly-goat anti-CD127 (R&D Systems) for 1 hour at room temperature, and then incubated with streptavidin-HRP. Reactions were developed with tetramethylbenzidine (TMB) substrate and results were obtained via a plate reader at 450 nm. Sample concentrations were extrapolated from standard curves of recombinant human CD127-Fc chimera, as previously described (169).

ELISPOT assays

NK cells (1×105) were plated with or without the presence of 10 ng/ml IL-7 (unless otherwise stated), IFNα (500 U/ml), or IL-12 (1 ng/ml) in ELISPOT plates pre-coated with anti-IFNγ (Human IFNγ mAb, clone 2G1,Thermo Scientific, Rockford, IL), TNF

(TNF3/4, MabTech) or Granzyme B (GB11, BioRad) capture mAb and cells were cultured 20 hours at 37°C. In some assays, IL-7-treated cells were cultured additionally with either wortmannin or P-STAT5 inhibitor as stated above. Secondary antibody was added and spots were detected as previously described (170).

CD107a degranulation assay

K562 cells (1 × 104) were seeded into 96-well round bottom plates overnight. Freshly purified bulk NK-cells were stained with anti-CD107a antibody (H4A3) or isotype control (BD Biosciences) at 37°C, 5% CO2 for 1 hour, supplemented with 4 µL of protein transport inhibitor (BD GolgiStop), then co-cultured with K562 cells at 5:1 E:T for 5 hours. Control cultures were performed with NK cells alone. NK cells were washed and stained with anti-CD3-14-19-AlexaFluor700 (UCHT1, M5E2, Biolegend), anti-

CD56-PE-Cy7 (B159, Biolegend), anti-CD16-APC-H7 (3G8, BD Biosciences).

NK cell cytolytic activity

Huh7.5 hepatoma cells were provided by Dr. C. M. Rice (Apath LLC). The pJFH1 plasmid was provided by Dr T. Wakita (Japan). Infectious JFH1 virus was prepared as described (171). Two thousand Huh7.5 cells were plated, and cultured for one day, and then infected with 1 multiplicity of infection (MOI) of JFH-1, 52 hours prior to NK cell co-culture.

NK cells were isolated from PBMCs within 3 hours of phlebotomy as described above, and tested for cytolytic activity against JFH-1–infected or uninfected Huh7.5 cells or

K562 cells, directly, or after pre-treatment with 10 ng/ml IL-7 (unless otherwise stated),

500 U/ml of IFN-α2a or complete RPMI medium for 16 hours and washed. 5 x 104 NK cells were added to HCV-infected or uninfected HuH 7.5 cells or K562 cells (10,000 at time of co-culture) to achieve an effector to target ratio (E:T) ratio of 5:1 for 5 hours.

NK-Huh7.5 cell co-culture supernatant was analyzed by M30 enzyme-linked

52 immunosorbent assay (DiaPharma), which detects Huh cell caspase-cleaved cytokeratin

18 (CK-18). NK-K562 cell co-culture supernatant was analyzed by LDH release assay.

Spontaneous effector and target, and maximal lytic reagent–induced target cell death was evaluated in control wells.

Statistical analysis

Statistical analyses were performed with GraphPad Prism software, version 5.04. We used Mann–Whitney U test for 2-way comparisons of continuous variables between groups. We used Wilcoxon Signed Rank related samples test for non-parametric comparison of related continuous variables within groups, and Spearman rank correlation coefficient to analyze associations between continuous variables. All tests of significance were two-sided and p values <0.05 were considered significant.

Results

CD56bright NK cell CD127 expression is negatively associated with plasma HCV level

We first analyzed cryopreserved PBMC in each study group (Table 1) for NK cell subset frequency, characterizing NK cells based on surface expression of CD56 and CD16 on lymphocyte gated cells that were viable and CD7+CD3-CD14-CD19- (Fig. 2.1a). There was no difference in total NK cell frequency or NK cell subset frequency comparing chronic HCV, HIV or HCV-HIV co-infected subjects with uninfected donors.

Since CD127 is the cellular receptor for IL-7 and also plays an important role in modulating IL-7 activity in plasma (126), we next measured CD127 expression on NK cell subsets in samples from each group. CD127 was most abundantly expressed on the

CD56bright NK subset (Fig. 2.1a), consistent with previous literature (136). Comparing groups, CD56bright NK subset CD127 expression was similar comparing HCV mono- infection to uninfected donors (Supplemental Fig. 2.1a). Notably however, the proportion of CD56bright NK cells expressing CD127, but not mean fluorescent intensity

(MFI) of CD127 expression, was negatively correlated with plasma HCV levels in HCV mono-infected subjects (r= -0.400, p= 0.020, Fig. 2.1b) and in HCV-HIV co-infected subjects (r= -0.340, p= 0.037, Fig. 2.1c). This negative relationship was also observed upon secondary analysis of purified NK cells from HCV mono-infected subjects (r= -

0.500, p= 0.030, Supplemental Fig. 2.1b).

Figure 2.1. CD56bright NK cell CD127 expression negatively associates with HCV level. (A- C) Cryopreserved PBMC from uninfected donors (UD), HCV, HIV and HCV-HIV infected subjects were measured for NK cell subset frequency and CD127 expression. FSC-SSC gating is applied first to identify lymphocyte populations, followed by a viability cell gate for live cells. NK cells are defined as CD7+3-14-19-56+/-16+/-. NK cell subsets classified by CD56 and CD16 expression. CD127 expression (black) is measured within each NK cell subset and compared to isotype (light gray), shown here from an uninfected donor (A). B-C: Negative correlation between HCV infected (B) and HCV-HIV co-infected subjects (C) CD56bright CD127 frequency and plasma HCV level. Serum IL-7 was measured from UD, HCV, HIV and HCV-HIV infected subjects by ELISA (D).

In addition to downstream signaling, IL-7 binding to CD127 can result in lower cell

surface CD127 expression (126). Whether the negative association between CD56bright

NK cell CD127 expression and HCV level was attributable to serum IL-7 levels was next

evaluated. Serum IL-7 was lower in HCV mono-infected subjects compared to uninfected

donors (UD) (p= 0.040, Fig. 2.1d), consistent with previous literature (172). In contrast,

HIV-infected subjects had greater serum levels of IL-7 than uninfected donors (p=

0.014), and HCV-HIV co-infected subjects had higher serum IL-7 levels than HCV

mono-infected subjects (p= 0.018, Fig. 2.1d). There was a trend towards an inverse

association between serum IL-7 level and CD4 count in HIV mono- and HCV-HIV co-

infected subjects (n=10, r= -0.576, p=0.088; n=9, r= -0.583, p=0.108, respectively),

consistent with prior literature (124). There was no association between serum IL-7 levels

and HCV levels or CD127 expression on CD56bright NK cells.

Supplemental Figure 2.1. CD56bright NK cell CD127 of purified NK cells expression is unaltered in HCV and HIV infection and negatively associates with HCV level. CD127 expression was measured by proportion of CD56bright NK cells (%) by flow cytometry (as in Figure 1) within purified NK cells (1x105) of UD (n=15), HCV (n=19), HIV (n=18) and HCV-HIV (n=12) infected subjects (A). Negative correlation between HCV infected subjects CD56bright CD127 frequency and plasma HCV level (B).

IL-7 induces CD56bright NK cell activation and this is impaired in HIV infection

Given that expression of CD127 on CD56bright NK cells correlated negatively with plasma

HCV level, we considered the possibility that IL-7 signaling plays a role in NK cell control of HCV infection. We therefore next sought to directly determine the effect of IL-

7 on NK cell activation in ex vivo assays. Purified NK cells of uninfected donors (n=20), uncontrolled HCV (n=20), virally suppressed HIV (n=16) and HIV-HCV co-infected

(n=13) subjects were cultured in the presence or absence of IL-7, IFNα or IL-12 overnight. CD69, NKp30, NKG2A and IFNaR1 expression was measured on each NK cell subset as indicators of NK cell activation and IFNα response capacity. Since the dose response relationship of IL-7 on NK cell activation and the expression of these surface markers was unknown, we carried out a concentration curve titration of IL-7 ranging from 0.01 ng/ml to 100 ng/ml. While there was no effect of IL-7 on NK cell subset expression of NKp30, NKG2A or IFNaR1, IL-7 induced CD56bright NK cell CD69 expression in a dose dependent manner (Supplemental Fig. 2.2a). At concentrations higher than 10 ng/ml, the effect of IL-7 on NK cell activation plateaued. At 10 ng/ml IL-

7, we observed significant enhancement of CD56bright NK cell CD69 expression in uninfected donors, measured both as the proportion of cells expressing CD69 and as MFI

(Fig. 2.2a-b, p= 0.020, 0.040, respectively). A similar pattern was observed with NK cells from HCV infected subjects (p= 0.008, p= 0.009; Supplemental Fig. 2.2b-c) but not HIV or HCV-HIV infected subjects. Additionally, both IL-12 and IFNα enhanced the expression of CD69 on CD56neg and CD56dim NK cells of uninfected donors (Fig. 2.2a) and HCV infected subjects. CD56neg, CD56dim and CD56bright NK cell CD69 upregulation in response to IFNα was impaired in HIV (n=7) and HIV-HCV (n=5) co-infected

57 subjects, indicating that HIV infection impairs both IL-7 and IFNα-mediated NK cell activation, while HCV does not. Upon co-stimulation with IL-7 and IFNα, we observed an additive effect of each cytokine on CD56bright NK cell CD69 expression in samples from uninfected donors (p= 0.020, Fig. 2.2a, c). This combined effect of IL-7 and IFNα was not above that induced by IFNα alone in HCV infected (n=7) and HIV infected subjects (n=6). Overall, IL-7 can activate CD56bright NK cells and enhance IFNα- mediated CD56bright NK cell activation as measured by CD69 upregulation. However, this

IL-7-mediated CD56bright NK cell CD69 upregulation is impaired in HIV and HCV-HIV co-infected subjects, and the combined effect of IFNα and IL-7 is not observed in either

HCV or HIV infection.

Figure 2.2. IL-7 activates CD56bright NK cells and enhances IFNα-mediated CD56bright NK cell activation via CD69 upregulation. Purified NK cells (1x105) of uninfected donors (UD, n=20) were plated in a round-bottom plate in presence or absence of 10ng/mL IL-7 and/or 500U/mL IFNα for 20hours, and CD7+3-14-19- CD56bright, CD56dim, and CD56neg NK cell subsets were assessed for CD69 surface expression. Here we show CD69 expression (%, MFI upper right corner) of the CD56bright, CD56dim and CD56neg NK cells subset of an uninfected donor (A), and the frequency (%, left) and MFI (right) of CD69 within the CD56bright NK cell subset of UD (n=20, B). IFNα- and IFNα + IL-7-mediated CD56bright NK CD69 MFI induction shown as level above medium alone conditions within UD (n=12, C).

Supplemental Figure 2.2. IL-7 upregulates CD56bright NK cell CD69 expression in dose response fashion and promotes CD56bright CD69 upregulation in HCV infected subjects. Purified NK cells (1x105) were plated in a round-bottom plate in presence or absence of increasing IL-7 (one representative UD, A) or 10ng/mL (B-C) as described in Figure 2. Here we show CD69 expression (%, middle; MFI, upper right corner) of the CD56bright NK cell subset of an HCV infected, HIV infected and HCV-HIV co-infected subject (B) and MFI of CD69 within the CD56bright NK cell subset of HCV infected (n=20), HIV infected (n=16) and HCV-HIV co-infected subjects (n=13, C).

IL-7 induces NK cell IFNγ production in uninfected, HCV and HIV infected subjects, and this ex vivo activity is associated with CD127 expression

Previous findings demonstrate that IL-7 can promote CD56bright NK cell IFNγ production in uninfected control subject samples and those of patients with multiple sclerosis (173).

Here, we extend these findings to determine the effect of IL-7 on NK cell IFNγ and

TNFα expression during chronic viral infection. IL-7 promoted purified NK cell IFNγ release, as measured by ELISPOT. This was observed to be in a dose-dependent fashion, with a dose response similar to the pattern seen for CD69 upregulation, plateauing at 10 ng/ml (Fig. 2.3a-b). We also observed an increase in NK cell TNFα release upon IL-7 stimulation of samples from HCV mono-infected subjects (p=0.030, not shown). IL-7 induced NK cell IFNγ release from samples of uninfected donors (n=20, p< 0.0001, Fig.

2.3c), and this effect was also observed in HCV (n=20, p< 0.0001), HIV (n=20, p=

0.0015) and HIV-HCV (n=15, p= 0.0005) infected subjects (Fig. 2.3c). Additionally, the magnitude of IL-7-induced IFNγ from NK cells did not differ among groups. In contrast, the IFNα- and IL-12-induced NK cell IFNγ release were impaired in samples from HCV infected, HIV infected, and HCV-HIV co-infected subjects (not shown), consistent with previous findings (174). Consistent with the additive effect of IL-7 and IFNα on

CD56bright NK cell CD69 upregulation, there was an additive induction of IFNγ upon NK cell culture with these two cytokines with samples from uninfected donors (p=0.04, Fig.

2.3d), but not in HCV infected or HIV infected subjects.

Figure 2.3. IL-7 enhances NK cell IFNγ secretion and IFNα-mediated NK cell IFNγ release. Purified NK cells (1x105) of uninfected donors (UD, n=20), HCV (n=20), HIV (n=20) and HCV-HIV (n=14) infected subjects were plated in pre-coated IFNγ ELISPOT plates in presence or absence of increasing concentrations of IL-7 (A-B) or 10ng/mL IL-7 (C) and/or 500U/mL IFNα (D) for 20hours, and IFNγ secretion was measured as spot- forming units (SFU). Here were show a representative ELIPSOT plate of one UD (A) and dose response curve of UD (n =5, B). IFNα- and IFNα + IL-7-mediated IFNγ secretion is shown as level above medium alone conditions in UD (n=12, D).

To evaluate whether IL-7 signaling may play a role in NK cell-mediated control of HCV, we next examined the baseline CD56bright NK cell CD127 expression in relation to IL-7- induced IFNγ. There was a positive correlation between CD127 expression and IL-7- mediated bulk NK cell IFNγ production from uninfected donors (r= 0.560, p= 0.030) and

HCV mono-infected subjects (r= 0.560, p= 0.020; not shown), but not in HIV infected or

HCV-HIV co-infected subjects. Additionally, we observed a weak negative trend towards a relationship between IL-7-induced NK cell IFNγ release and HCV level in HCV mono- infected subjects (r= -0.370, p= 0.120; not shown), supporting a possible role for IL-7- dependent NK cell IFNγ in control of HCV. To confirm the IL-7-induced NK cell IFNγ release, we analyzed NK cell subset intracellular IFNγ expression, and found that IL-7, at

10 ng/ml, indeed induced CD56bright NK cell intracellular IFNγ production in uninfected donor subjects, and this remained intact in samples from HCV, HIV and HIV-HCV infected subjects (not shown).

IL-7-dependent NK cell survival and cell cycling are impaired in HCV and HIV infections

Previous findings have shown that IL-7 promotes CD56bright NK cell survival via upregulation of Bcl2 (136) and induces cell cycling (173). Whether this activity is altered during chronic viral infection is unknown. We first examined the effect of IL-7 on NK cell subset proportions over the time course of purified NK cell cultures. IL-7 did not alter the overall expression of CD56, as measured by MFI, in CD7+3-14-19- flow gated, bead purified NK cells of uninfected donors. IL-7 treatment increased the proportion of

NK cells in the CD56bright NK cell subset (p=0.008), and a trend towards reduced

63 proportions of CD56dim NK cells (p=0.07, not shown) in overnight cultures. There was no effect of IL-7 on CD56neg NK subset proportion in these same cultures. In HIV- infected and HCV-HIV co-infected subjects, there was a similar effect of IL-7 on the proportion of the CD56bright NK cell subset (p=0.041, p=0.042, respectively) in overnight cultures. The effect of IL-7 on NK cell subset distribution in overnight cultures was muted in the presence of HCV infection, with no increase in the proportion of NK cells within the CD56bright NK cells nor any effect on the CD56dim or CD56neg NK cell subsets.

To examine cell cycling and resistance to cell death, freshly obtained PBMC from uninfected donors (n=12), HCV (n=11), HIV (n=12) and HIV-HCV (n=8) co-infected subjects were cultured in the presence or absence of IL-7 (10 ng/ml) overnight. The next day, we performed intracellular staining for Bcl2 and Ki67. IL-7 induced Bcl2 and Ki67 expression in CD56bright NK cells of uninfected donors (Fig. 2.4a), consistent with previous literature (136, 173), but not in the CD56dim or CD56neg NK cell subsets. IL-7- induced CD56bright NK cell Bcl2 expression tended to be lower in HCV mono-infected subjects (p= 0.070 Fig. 2.4b), and IL-7-induced cycling (Ki67 expression) was lower in

HCV, HIV and HIV-HCV infected subject samples (p= 0.070, p= 0.030, p= 0.004, respectively compared to uninfected donors Fig. 2.4c). These results indicate that IL-7- induced NK cell cycling is impaired in direct ex vivo analysis of samples from HCV and

HIV infected persons.

Figure 2.4. IL-7-promotion of CD56bright NK cell Bcl-2 expression and cycling is impaired in HCV and HIV infections. PBMC (1x106) were plated in a round-bottom 96 well plate in presence or absence of IL-7 (10ng/mL) for 20 hours. Lymphocytes were assessed by forward and side scatter and NK cells were characterized by expression of CD7+3-14-19- and surface expression of CD56 and CD16. Here we show Bcl-2 (A, left) expression as MFI and Ki67 expression (A, right) as frequency (%) within CD56bright NK cells. IL-7-mediated CD56bright NK cell Bcl-2 MFI upregulation (B) and Ki67 induction (C) shown as fold change within controls (n=12), HCV (n=11), HIV (n=12) and HIV-HCV (n=8) subjects. Mean +/- SEM shown.

IL-7-induced NK cell degranulation is intact during HCV and HIV infection, but IL-7

enhanced NK cell killing of K562 targets is impaired

Cytotoxicity is a vital anti-viral NK cell effector function which can occur through

multiple mechanisms. Previous studies have shown that IL-7 can enhance CD56bright NK

cell CD107a expression (173). We therefore measured NK cell subset upregulation of

CD107a, a degranulation marker, in purified NK cells after culture with K562 target

cells, in the presence or absence of IL-7. In samples from uninfected donors, we observed

significant IL-7-mediated enhancement of CD56bright NK cell CD107a expression above

that induced by K562 target cells (n=10, p= 0.0004, Fig. 2.5a-b), while there was no

effect of IL-7 on CD107a expression of the CD56dim or CD56neg NK cell subsets (Fig.

2.5a). This enhancement of CD56bright NK cell CD107a expression was also seen in

samples from HCV infected subjects (n=10, p= 0.0137) as well as HIV infected subjects

(n=7, p=0.016; Fig. 2.5a). In a limited analysis of a small number of HCV-HIV co-

65 infected subjects (n=3), we observed a similar pattern (Fig. 2.5a). Comparing the magnitude of IL-7-induced CD56bright NK cell CD107a expression, we observed no significant differences among groups, indicating that IL-7-mediated NK cell degranulation is likely intact in both chronic viral infections.

In order to further determine the ability of IL-7 to promote NK cell cytotoxicity, we analyzed purified NK cell granzyme B release using ELISPOT. Upon overnight culture, we observed IL-7 induced NK cell granzyme B release in samples from uninfected donors (n=18, p= 0.0008) and HCV infected subjects (n=17, p= 0.0004, not shown).

However, this IL-7-induced NK cell granzyme B release was impaired in samples from

HIV infected and HIV-HCV co-infected subjects (not shown).

TRAIL is known to mediate cytotoxicity (122). We therefore next evaluated the ability of

IL-7 and IFNα to induce NK cell subset surface expression of TRAIL after overnight stimulation. In all subject groups, there was no effect of IL-7 on CD56dim or CD56neg NK cell TRAIL expression, while there was a robust effect of IFNα on TRAIL expression in the same NK cell subsets (Fig. 2.5c). In contrast, with samples both from uninfected donors and HCV infected subjects, there was a significant increase of CD56bright NK cell

TRAIL expression upon IL-7 stimulation (n=10, p= 0.008; n=10, p=0.008, Fig. 2.5c).

The magnitude of IL-7-induced TRAIL expression was significantly lower than that induced by IFNα in uninfected donors (n=10, p= 0.030, Fig. 2.5c). In a smaller sample set of HIV infected (n=5) subjects, we saw a similar pattern, with moderate IL-7-induced

CD56bright NK cell TRAIL expression (p=0.125 for HIV group), also with greater TRAIL induction upon culture with IFNα (Fig. 2.5c). Huh7.5 target cells have been shown to be lysed by NK cells via IFNα-induced TRAIL (122). To understand if IL-7 facilitates NK cell cytotoxicity via TRAIL, we cultured purified bulk NK cells pre-treated with IL-7 or

IFNα with Huh7.5 target cells with or without HCV JFH-1infection. IL-7 did not induce bulk NK cell lysis of JFH-1 infected Huh7.5 cells, whereas NK cells stimulated with

IFNα did tend to induce cytolysis of JFH1 infected Huh 7.5 cells (n=5, p=0.156,

Supplemental Fig. 2.3). Together, these results indicate that while IL-7 increases

CD56bright NK cell TRAIL expression, the enhanced expression is not sufficient to promote TRAIL-dependent cytotoxicity in the in vitro system used here.

Supplemental Figure 2.3. JFH-1 infected Huh7.5 cell lysis in response to IL-7 and IFNα. Purified NK cells (1x105) were plated in a round-bottom plate in presence or absence of IL-7 (10ng/ml) or IFNα (500U/mL) over night. NK cells were then washed and cultured for 5 hours in presence of JFH-1 infected Huh7.5 cells. NK cell cytolysis of Huh7.5 cells was measured by M30 assay with UD (media and IL-7, n=10; IFNα, n=5).

Notably, a previous report has shown that IL-7 promotes NK cell cytolytic activity via

FasL-Fas interactions (102). We did observe an increase in FasL (CD95L) expression in

CD56bright NK cells upon IL-7 stimulation in both uninfected donors and HCV infected subject samples (n=10, p= 0.008; n=10, p=0.078, Fig. 2.5d). There were similar patterns in a smaller number of HIV infected subject samples (n=5, Fig. 2.5d). In contrast, there was no significant increase in FasL expression in CD56dim or CD56neg NK cells upon IL-

7 treatment (Fig. 2.5d). IFNα stimulation also enhanced CD56bright NK cell FasL expression in samples from uninfected donors (n=6, p=0.030). In contrast to the effects on TRAIL expression, there was no significant difference in magnitude of IL-7- and

IFNα-mediated FasL upregulation in any of the subject groups (Fig. 2.5d), nor any difference in IL-7- or IFNα-induction of FasL on NK cells across subject groups. Next, we cultured NK cells with K562 cells, which we confirmed were susceptible to FasL- mediated cytolysis using a FasL agonist (not shown). Indeed, NK cell lysis of K562 target cells was enhanced in presence of IL-7 (n=10, p= 0.039, Fig. 2.5e). The data here suggest that IL-7-mediated enhancement of NK cell lysis of K562 target cells may be impaired in samples from HCV infected, HIV infected and HIV-HCV co-infected subjects (p=0.11, Fig. 2.5f).

Figure 2.5. IL-7 promotes NK cell cytotoxic function, which is impaired in HIV and HCV infections. Purified NK cells (1x105) were cultured in presence or absence of IL-7 (10ng/mL) for 20hours. NK cells of UD (n=10), HCV (n=10), HIV (n=8) and HCV-HIV (n=3) were then cultured with K562 cells for 5 hours (A-B, E-F). CD7+3-14-19-CD56bright, CD56dim and CD56neg NK cells were assessed for CD107a expression by flow cytometry (A-B). In NK cell monocultures without K562 stimulation, no CD107a expression was observed. Representative data from one UD (A). CD7+3-14-19- CD56bright NK cell TRAIL (C) and FasL (D) surface expression was measured by flow cytometry (representative histogram flow plot showing each NK cell subset of one UD); UD n=8, HCV n=9. NK cell cytolysis of K562 cells was measured by LDH assay with UD (n=10) (E). UD, (n=10), HCV (n=10), HIV (6) and HCV-HIV (n=2); results depicted as frequency of K562 lysis above medium alone conditions (F). Mean +/- SEM shown.

IL-7-induced NK cell signaling is impaired in chronic HCV and HIV infection, yet restored after HCV clearance.

The impairments in IL-7-mediated NK cell survival, activation and cytolytic activity in

HCV and HIV infections may be attributable to changes in IL-7 signaling. We therefore next inquired if there were overall impairments in proximal IL-7 signaling in these chronic viral infections. IL-7 signaling requires internalization of the receptor (126). We therefore first analyzed NK cell subset cell surface CD127 expression after overnight culture with IL-7 (Fig. 2.6a). Compared to uninfected donors (n=16), we observed significantly less downregulation of CD127 surface expression on the CD56bright NK cell subset in response to IL-7 stimulation in HCV infected (n=18, p= 0.02), HIV infected

(n=13, p= 0.007) and HIV-HCV co-infected (n=12, p= 0.009) subject samples (Fig.

2.6b).

After CD127 internalization, IL-7R signaling leads to phosphorylation of STAT-5

(pSTAT-5), which has been shown to be impaired in T cells of HIV infected subjects

(175). We hypothesized that the impairments in IL-7-mediated NK cell activities in these infected subjects were due to attenuated IL-7 signaling. At 15 minutes of IL-7 stimulation, pSTAT-5 increased in CD56bright NK cells of uninfected donors, and this did not occur in HCV, HIV infected or HIV-HCV co-infected subjects (not shown).

Furthermore, phosphorylation of STAT-5 in CD56bright NK cells in response to one day of

IL-7 stimulation was impaired in HCV infected, HIV infected and HCV-HIV co-infected subjects (Fig. 2.6c).

Figure 2.6. IL-7/CD127 signaling is impaired in chronic viral infection. PBMC (1x106) (C-D) or purified NK cells (A-B, 1x105) were plated in round-bottom 96 well plate in presence or absence of IL-7 (10ng/mL). Lymphocytes were assessed by forward and side scatter and NK cells were characterized by expression of CD7+3-14-19- and surface expression of CD56 and CD16. CD7+3-14-19- CD56bright NK cell CD127 MFI expression was measured after 20hrs (representative plot of one UD shown, compared to isotype, A), represented as level above medium alone conditions (B); UD (n=16), HCV (n=18), HIV (n=13), HCV-HIV (n=12). (C-D) CD56bright NK cell pSTAT-5 induction measured as foldchange compared to medium alone treatment within UD (n=10), HCV (n=11), HIV (n=9) and HIV-HCV (n=9) subjects (C). IL-7-induced CD56bright NK cell pSTAT-5 expression (above medium alone conditions) before and after achieving sustained virologic response upon HCV therapy (D). Mean +/- SEM shown.

Because IL-7-mediated NK cell IFNγ release remained intact in infected subjects (Fig.

2.3c), we next investigated the extent to which CD127 signaling relied on another transcription factor, AKT. To this end, IL-7-induced NK cell IFNγ release was assessed by ELISPOT in the presence or absence of STAT-5 or AKT inhibitors. Interestingly, inhibition of either STAT-5 or AKT impaired IL-7-induced NK cell IFNγ release

(Supplemental Fig. 2.4). This suggests that IL-7 induced NK cell IFNγ release relies on both STAT-5 and AKT, and offers potential insight as to why IL-7-mediated NK cell

IFNγ function remains intact while IL-7-signaling, as measured by pSTAT-5 induction, is impaired in chronic HCV and HIV infection.

Supplemental Figure 2.4 IL-7-mediated NK IFNγ release is dependent on AKT and STAT-5. Purified NK cells (1x105) of uninfected donors (n=3) were plated in pre-coated IFNγ ELISPOT plates in presence or absence of 10ng/mL IL-7, as in Figure 3, with or without AKT-inhibitor (wortmannin) or STAT-5-inhibitor.

Previous work has demonstrated that the soluble form of CD127 (sCD127), is elevated in serum of HIV infected donors, and this inhibits IL-7 activity (169). sCD127 was measured in serum of uninfected donors (n=5), HCV infected (n=5), HIV infected (n=5) and HCV-HIV (n=5) co-infected subjects, and significantly elevated levels found in

HCV-HIV co-infected subjects (p=0.02, not shown). To determine the durability of the effect of HCV infection on NK cell CD127 signaling, we compared IL-7-mediated

CD56bright NK cell STAT-5 phosphorylation in HCV infected subjects before and after achieving sustained virologic response (SVR) to IFN-free direct-acting antiviral therapy.

Whereas there was no observed IL-7 induced STAT-5 phosphorylation prior to therapy, there was IL-7 induced STAT-5 phosphorylation six months after institution of therapy and clearance of the virus, (Fig. 2.6d) that was similar in magnitude to that observed in uninfected donors (Fig. 2.6c). This is consistent with a reversible effect of HCV on proximal IL-7 signaling.

Discussion

CD127 is most abundantly expressed on the CD56bright subset of NK cells (136) (Fig.

2.1a), which is a dominant NK cell subset of secondary lymphoid tissue and liver (111).

CD56bright NK cells are potent cytokine producers and pivotal in dendritic cell maturation, central to effective T cell priming (112, 115). Understanding the biology of this subset is needed to advance our understanding of factors involved in HCV pathogenesis.

Investigations by others have demonstrated a negative relationship between CD8 T-cell

CD127 expression and plasma HCV level (134, 135). Here, for the first time, we show how CD56bright NK cell CD127 expression is negatively associated with plasma HCV level in both HCV mono-infection and HIV-HCV co-infection (Fig. 2.1b-c). This negative correlation could be interpreted as indication that HCV infection may be exerting a negative influence on CD127 expression. However, CD56bright NK cell CD127 was not lowered in HCV mono-infected or HCV-HIV co-infected subjects compared to uninfected donors (Supplemental Fig. 1a). Alternatively, CD127 expression may determine NK cell effector function contributing to control of HCV.

Importantly in the case of viral infection, IL-7 supports a strong Th1-type immune response by induction of IFNγ and IL-2 production (124). CD8 T-cell CD127 expression is positively associated with IFNγ-producing capacity in HCV infected subjects (134,

135). IL-7 has also been shown to augment CD56bright NK cell degranulation and IFNγ production, with heightened effects in NK cells of multiple sclerosis patients (173). In both HCV and HIV infection, NK cells have been observed to have reduced IFNγ producing capacity compared to those of uninfected donors, including in response to IL-

12 and IL-15 (92, 97, 121, 123, 176). These effector functions are thought to be essential to effectively clear HCV from the infected host (89, 92). Similarly, NK cells of subjects who naturally control HIV produce greater amounts of IFNγ than AIDS progressors and uninfected donors in direct ex vivo assays (177). Here, we show that IL-7 enhances NK cell IFNγ release, and demonstrate IL-7-mediated NK cell IFNγ release and production is intact within the setting of HCV and HIV infection (Fig. 2.3c). Furthermore, we observed a positive association between baseline CD56bright NK cell CD127 expression and IL-7- mediated NK cell IFNγ release in HCV mono-infected subjects (r= 0.560, p= 0.020, not shown), as well as positive trends between CD56bright NK cell CD127 expression and IL-

7-mediated CD69 upregulation within uninfected donors (r= 0.481, p=0.084) and HCV mono-infected subjects (r= 0.416, p=0.086). We also found a trend towards a negative correlation between IL-7-mediated NK cell IFNγ release and plasma HCV level (r= -

0.370, p= 0.120, not shown) in HCV mono-infected subjects. Together, these data may be consistent with IL-7 induced, CD127 dependent, NK cell IFNγ release playing a role in host control of HCV.

Our data demonstrate further that many IL-7 mediated effects on NK cells are impaired in

HCV and HIV infected subjects. While a previous study showed that IL-7 induced Bcl-2 expression in CD56bright NK cells in uninfected donors (Fig. 2.4a) (136), we demonstrate here that IL-7 induced Bcl-2 expression in CD56bright NK cells is impaired in HCV infection (Fig. 2.4b). The IL-7-enhanced Ki-67 expression in this subset was also impaired in HCV, HIV and HIV-HCV co-infected subject samples (Fig. 2.4c). One potential mechanism by which these viruses may be evading host response is by

75 suppressing IL-7-mediated NK cell survival and cell cycling. While IL-7-mediated

CD56bright NK cell CD107a induction appeared to be intact in all virally-infected groups, we observed a lack of IL-7-mediated CD56bright NK cell CD69 upregulation

(Supplemental Fig. 2b-c) and granzyme B release in HIV infected and HIV-HCV co- infected subjects. Finally, the presence of both HCV and HIV infection had a negative effect on IL-7-mediated NK cell cytolysis of K562 target cells (Fig. 2.5f). We did not see any impairment with IL-7-mediated bulk NK cell granzyme B release or CD56bright NK cell FasL upregulation in HCV infected subjects (Fig. 2.5b and 2.5d, respectively). The overall impairment of IL-7-mediated NK cytolytic activity directed at K562 targets (Fig.

2.5f) may be in part attributable to the observed impairment in IL-7-induced Bcl-2 upregulation and cell cycling (Fig. 2.4b-c). By inhibiting the pro-survival and potential proliferative effect of IL-7 signaling on CD56bright NK cells, HCV may limit the available pool of NK cells capable of IL-7-mediated cytolytic activity. These data suggest that regulation of other NK cell receptors and activity than CD107a induction is involved in

IL-7-mediated NK cell cytolytic activity.

In HIV infection, it is known that T-cell IL-7 signaling is impaired at the level of pSTAT-

5 (175, 178), the major transcription factor through which IL-7 signals. Here, we saw a similar pattern of impaired pSTAT-5 induction in CD56bright NK cells of HIV infected and HIV-HCV co-infected subjects (Fig. 2.6c). We also observed a similar impairment in IL-7-mediatied pSTAT-5 induction in HCV mono-infected subjects (Fig. 2.6c). While

IL-7/CD127 signals through STAT-5, IL-7 also signals through AKT [27]. In a small sample set of uninfected donors (n=3), we evaluated the effect of blocking AKT vs.

STAT-5 on IL-7-mediated NK cell functions. Inhibition of STAT-5 led to impaired IL-7- mediated CD56bright NK cell Bcl2 enhancement as well as reduced TRAIL induction (not shown), supporting previous work in mice which indicated that STAT-5p is necessary for

NK cell proliferation and lytic activity (179). When AKT was blocked, IL-7-induced

CD56bright NK cell FasL expression was minimized. Inhibition of STAT-5 or AKT impaired IL-7 mediated NK cell IFNγ release (Supplemental Fig. 2.4), suggesting that although STAT-5 signaling is impaired in HIV and HCV infection, perhaps IL-7- mediated AKT signaling is sufficiently intact to promote NK cell IFNγ release and production.

Another factor that may be contributing to the observed impairments in IL-7-mediated

NK cell activity within HIV and HCV infection is reduced CD127 downregulation/internalization upon IL-7 ligation (Fig. 2.6b), supported by previous findings (175, 180). In HCV mono-infection, we observed significantly lower levels of serum IL-7 compared to uninfected donors (Fig. 2.1e), indicating that HCV infection may also impair IL-7-mediated signaling via reduction of available IL-7 (173), in contrast to what is observed in HIV infection (Fig. 2.1d). This is consistent with previous findings that have shown reduced levels of serum IL-7 (172) and hepatic IL-7 in chronic HCV infected subjects (181), and may reflect impaired hepatic IL-7 producing function. In support of the latter, we did observe a trend towards a positive association between serum

IL-7 and platelet count in HCV infected subjects (r=0.340, p=0.120). When HCV infected subjects achieved SVR, CD56bright NK cell IL-7-mediated STAT-5 phosphorylation improved (Fig. 2.6d), suggesting that HCV infection more likely has a

77 relatively short term reversible negative impact on the IL-7/CD127-mediated NK cell

STAT-5 signaling pathway.

CD127 haplotype studies have indicated the important role of the IL-7Rα in immune cell function. Notably, these haplotype studies include the finding of associations with IL-

7Rα polymorphism rs6897932 C/T; while the T allele was shown to be protective from multiple sclerosis, the C allele positively associated with the risk of multiple sclerosis

(182, 183). The rs6897932 CC genotype has also been associated with severe liver disease in HIV-HCV co-infected subjects (184). The rs6897932 C allele leads to increased formation of sCD127 (184), and sCD127 has also been shown to be elevated in

HIV infection (169). sCD127 competes with its membrane-bound counterpart for IL-7, leading to elevated levels of CD127 within T cells (169). Here, we observed a trend for enhanced plasma levels of sCD127 in both HCV and HIV mono-infections compared to uninfected donors, and significant enhancement in HCV-HIV co-infection. Furthermore, soluble mediators of immune activation, which are elevated in HIV infection, including

IL-6, have been shown to impair CD4+ T cell IL-7 signaling (178). Taken together, we can infer that both HCV and HIV have overlapping, yet distinct mechanisms of impairing

IL-7-mediated NK cell signaling and anti-viral immune effector functions.

While effective new HCV therapy exists, treatment-resistant populations remain (67), and

HCV eradication altogether worldwide will most likely require a preventative vaccine.

Further evaluation of the role of NK cell anti-viral effector functions will guide

78 immunotherapeutic strategies to improve viral control and vaccine designs. Finally, further studies are warranted to elucidate the mechanisms by which these chronic viral infections dampen the effect of IL-7 on NK cells.

Chapter 3:

bright - CD14 CD16 monocytes and sCD14 level negatively associate with CD4-memory

T-cell frequency and predict HCV-decline on therapy

*This article was originally published in Journal of Acquired Immunodeficiency Syndromes. Chelsey J. Judge, Johan K. Sandberg, Nicholas T. Funderburg, Kenneth E. Sherman, Adeel A. Butt, Minghee Kang, Alan L. Landay, Michael M. Lederman, Donald D. Anthony (2016). CD14brightCD16- monocytes and sCD14 level negatively associate with CD4-memory T-cell frequency and predict HCV-decline on therapy. J AIDS. June 1 10.1097/QAI.0000000000001104.

Abstract

During HIV-HCV co-infection CD14brightCD16--monocytes produce soluble immune- activation markers that predict disease-progression and poor IFNα-treatment response.

We evaluated relationships among immune-activation, monocyte phenotype, CD4- memory T-cells and HCV-, CMV- and CMV/EBV/Influenza (CEF)-specific IFNγ- response, before and during IFNα-treatment. Effector-memory and central-memory CD4-

T-cell frequencies were lower in HCV+HIV+ than uninfected-donors, and correlated negatively with HCV-level, CD14brightCD16--monocytes and plasma sCD14. sCD14 and

CD14brightCD16- monocytes negatively correlated with IFNα-dependent HCV-decline. sCD14 negatively associated and CD4 effector-memory T-cells positively-associated with CEF-specific IFNγ-response. These data support a role for memory-CD4 T-cells in

HCV-containment, and link immune-activation and CD14brightCD16--monocyte frequency to failure of interferon-dependent HCV-clearance.

Introduction

Immune-activation predicts morbidity during HIV-infection (185, 186). Soluble (s)

CD14, IL-6, IP-10 and sCD163 are plasma markers of immune-activation in chronic viral-infection (148, 151, 153, 186-189). Elevated sCD14 levels negatively predict response to HCV IFN-therapy during HCV-HIV co-infection (148, 151, 152). Monocyte- activation is also negatively associated with response to HCV-therapy (190), and monocytes contribute to immune-activation via mechanisms that include elaboration of sCD14, IP-10, sCD163 and IL-6 in HIV and HCV infections (191-194).

Containment and clearance of HCV is dependent on CD4 T-cells (70, 80, 195), and positively associated with an IFNγ and cytolytic function (70, 196-199). HCV-specific memory T-cells are positively associated with resolution of acute HCV-infection (195,

200). Monocytes may partly shape this HCV-directed response, via direct-contact and polarizing cytokines (198, 199). Our prior findings indicate soluble-factors of immune- activation, including IL-6 and sCD14, can impair CD4 T-cell responses (178, 201). We extend these studies here to evaluate the role of immune-activation, monocyte-subset frequency, and CD4-memory T-cells in host control of HCV and IFNα-treatment-induced

HCV-clearance during HCV-HIV co-infection.

Methods

AIDS Clinical Trials Group (ACTG) A5294 was a phase-3 trial evaluating efficacy of boceprevir/pegylated-interferon (PegIFN)/ribavirin (rbv) in the setting of HCV-HIV co- infection. PegIFNα/rbv lead-in for 4 weeks preceded addition of boceprevir. Sixty-four

HCV-treatment-naïve participants from A5294 whose entry criteria included at least 8 weeks on antiretroviral-therapy (ART) with CD4 count >200/mm3, genotype 1 HCV- infection, and HIV-1 RNA <50 copies/mL and HCV RNA data were selected. After obtaining IRB-approved consent, peripheral blood mononuclear cell (PBMC) and plasma samples were prepared at each clinical-site, cryopreserved, and sent to a central-storage facility. Cryopreserved PBMCs from baseline (week 0), and plasma from baseline and week 4 of boceprevir/PegIFNα/rbv treatment were analyzed. Plasma and PBMC from uninfected control (n=25), and HCV+ mono-infected (n=34) participants were obtained at the Cleveland VA hospital under a separate IRB-approved protocol.

Plasma was evaluated for sCD14, IL-6, IP-10 and sCD163 by enzyme-linked immunosorbent assay (R&D Systems, Minneapolis, Minnesota).

Cryopreserved PBMCs were thawed and stained with monoclonal antibodies. Viable lymphocyte-gated cells that were CD3+CD8-CD4+CD27-/+CD45RO-/+ were analyzed.

For monocyte phenotype, we quantified proportions of viable-gated cells that were

CD14bright/dimCD16-/+. CD86 expression was recorded as mean fluorescence intensity, compared to isotype control.

HCV-peptides (n=441, 18aa each, overlapping by 11aa) representing the entire HCV-1a

H77 sequence, CMV/EBV/Influenza (CEF) peptide pool (32 immunodominant CD8 epitopes) and CMV (pool of 138 peptides (15-mers) of the pp65 protein, overlapping by

11 aa) were supplied by the National Institutes of Health AIDS Research and Reference

Reagent Program (Division of AIDS, NIAID). HCV peptides were pooled together into

10 pools (27–61 peptides/pool) according to viral protein region (core peptides 1–27; E1 peptides 28–55; E2 peptides 56–107; NS2 plus P7 peptides 108–147; NS3-1 peptides

148–193; NS3-2 peptides 194–239; NS4 peptides 240–287; NS5A peptides 288–348;

NS5B-1 peptides 349–394; and NS5B-2 peptides 395–441). Peptide-pools were utilized at a final concentration of 2.7μg/mL each peptide (⩽0.5% DMSO; Sigma).

PBMCs (3×105 /well) were plated onto 96-well IFN-γ ELISPOT plates in presence or absence of CEF-, CMV-peptide, or each of the 10 HCV-peptide pools, incubated for

20hrs at 37°C, developed and analyzed as described (170, 202-205). A response was characterized as IFN-γ-production frequency 3-fold greater than mean-background frequency with ⩾15 spot-forming units (sfu) per well, as described (206). Using similar criteria, we have previously observed no responses to HCV-peptide pools in healthy control or disease control subjects (170, 202-205, 207).

We evaluated associations between continuous variables using Spearman rank correlation coefficients and inter-group comparisons by Mann-Whitney U test, both rank-based methods. All tests of significance were two-sided and P-values ≤ .05 were considered

84 significant. Analyses were performed using SPSS for Windows v. 20.0 (IBM Corp,

Armonk, New York).

Supplemental Table 3.1: Clinical Information. p- Variable HIV+HCV+ HCV (Genotype-1) Controls values

N 64 34 25

Age, years 47 (24-64) 58 (51-65) 53 (23-81)

Male sex, % 81 97 94

Race:

Black, % 47 47 56

White, % 41 50 32

other, % 13 3 12

Plasma HIV RNA level, Undetectable copies/mL

CD4 Count 677 (218-1530)

6 3 6 4 Plasma HCV 9.530 x 10 (5.730 x 10 - 1.500 x 10 (6.020 x 10 - 7 6 <0.001 level, IU/mL 7.260 x 10 ) 4.400 x 10 )

Serum 4.100 <0.001 albumin level, 4.300 (3.000-5.100) 3.900 (2.800-4.500) (3.500- g/dL 4.500) Platelet count, 203 (93-394) 223 (56–342) 227 (152- 0.200 3 k/cmm 313)

Serum AST 60 (19-172) 56 (20-243) level, UL 27 (16-47) <0.001

Serum ALT 75 (19-239) 64 (20–303) level, UL 28 (9-56) <0.001

APRI, 0.280 <0.001 AST/PLT 0.730 (0.210-2.250) 0.750 (0.160-3.550) (0.120- index 0.390)

Results

Baseline characteristics of the study participants are summarized in Supplemental Table

1. HCV+HIV+ co-infected participants had higher AST and ALT levels, higher and

APRI score than uninfected controls.

CD4 T-cell subsets were defined by expression of CD27 and CD45RO, with central- memory (CM) CD27+CD45RO+ and effector-memory (EM) CD27-CD45R0+, as previously described (208). The data demonstrate that CD4CM and EM frequencies were lower in HCV+HIV+ co-infected compared to uninfected participants and HCV+ mono- infected participants; while HCV mono-infected participants had higher CD4CM and

EM-T-cell frequencies compared to uninfected participants (Fig. 3.1a-b). While it is well documented that T-cell-mediated immunity is vital for HCV-clearance (70, 195-197,

200). The relationship between frequencies of CD4-memory T-cell subsets and HCV- control during HCV+HIV+ co-infection has not been investigated. We observed negative correlations between CD4CM T-cell frequency and HCV-level (r= -0.320, p=0.012), as well as CD4EM frequency and HCV-level (r= -0.368, p=0.004) prior to HCV-therapy in

HCV+HIV+ co-infected patients (Fig. 3.1c). We noted similar negative associations between CD8CM and EM-T-cell frequencies and HCV-level prior to start of HCV- therapy (r= -0.268, p= 0.039; r= -0.368, p= 0.004 respectively, not shown). CD4CM and

EM-T-cell absolute counts also negatively correlated with HCV-level (r= -0.273, p=

0.033; r= -0.33, p= 0.008, respectively, not shown). In HCV mono-infection, there was no association between CD4CM and EM frequencies and pre-treatment HCV viral load.

These results are consistent with CD4CM and EM T-cells playing a role in HCV-control during HCV-HIV co-infection.

To understand potential relationships among monocytes, HCV-level and CD4-memory T- cell populations, classical CD14brightCD16-, intermediate CD14brightCD16+ and patrolling

CD14dimCD16+ monocytes) were analyzed as described (157). In HCV-HIV co-infected participants, classical-monocyte frequency negatively correlated with CD4CM and EM frequencies (Fig 3.1d)and absolute counts (r= -0.609, p< 0.001; r= -0.478, p< 0.001, respectively, not shown).

Figure 3.1. CD4CM and EM Cells are reduced in HCV-HIV co-infection and negatively correlate with HCV-level and classical monocyte frequency. A) Representative CD4 T cell subset gating strategy of cryopreserved PBMC of HCV- HIV co-infected, HCV-infected and uninfected subjects (controls) which were stained with Yellow Live/Dead stain (Invitrogen, Grand Island, New York), anti-CD3- AlexaFluor700 (clone UCHT1), anti-CD14-Alexaflour700 (M5E2), anti-CD16-APC- H7 (3G8), anti-CD4-PE (RPA-T4), anti-CD8-PerCP (53-6.7), anti-CD27-PE-Cy7(M- T271), anti-CD45RO-FITC (UCHL1), anti-CD86-PE-Cy7 (IT2.2) or isotype controls. Flow cytometry data were acquired on a BD LSRII flow cytometer (BD Biosciences), and analyzed using FlowJo (TreeStar). Live cells were identified by forward and side scatter and viability. B) Week 0 CD4+CM and CD4+EM T cell frequencies (%) of each group. C) Baseline (Week 0) CD4 CM (top) and EM (bottom) frequencies of HCV-HIV co-infected participants in relation to HCV level in absence of exogenous IFN. D) Week 0 classical monocyte (CD14brightCD16-) frequency (%) of HCV+HIV+co-infected participants (n=49) in relation to CD4 CM and EM frequencies.

Upon activation, monocytes produce soluble markers of immune-activation, including sCD14, IP-10, IL-6 and sCD163 (191), known to be elevated in patients with chronic

HCV and HIV-infections (191-194). Expectedly, serum sCD163, IP-10, and IL-6 were significantly higher in HCV+HIV+ co-infected participants compared to uninfected controls (not shown). These soluble immune-activation markers positively correlated with each other. Specifically, IL-6 levels were positively correlated with both sCD163 and sCD14 levels (Supp Fig 3.1a-b), and sCD163 directly correlated with IP-10 levels

(Supp Fig 3.1c). sCD163 and IP-10 positively correlated with APRI score (r=0.571, p<0.001; r=0.300, p=0.017, respectively), a marker of liver fibrosis (Supp Fig 3.1d-e).

There was a trend towards classical-monocyte frequency positively correlating with levels of IP-10 (r=0.281, p=0.053), IL-6 (r=0.481, p=0.055), and sCD14 (r= 0.214, p=0.100) in HCV-HIV co-infected participants. No relationship was observed between classical-monocyte frequency and soluble markers in uninfected controls.

Supplemental Figure 3.1. Soluble markers of immune activation positively correlate with each other and liver damage score. A-E) Week 0 plasma IL-6, sCD163, sCD14 and IP-10 of HCV+HIV+co-infected participants (n=64) were measured by ELISA. A) IL-6 (pg/mL) levels in relation to sCD163 (ng/mL) B) IL-6 in relation to sCD14 (ng/mL) C) IP-10 (pg/mL) levels in relation to sCD163 D-E) APRI score in relation to sCD163 (D) and IP10 (E) levels.

Upon treatment with Peg-IFNα/rbv, we observed an increase in sCD14, similar to our previous findings (148), and an increase in IL-6 during 4 weeks of therapy (not shown).

In contrast, levels of sCD163 and IP-10 decreased during treatment (not shown). Baseline classical-monocyte frequency correlated negatively with HCV-decline at week 4 of

PegIFNα/rbv-therapy (r=-0.33, p=0.020, Fig. 3.2a). We found a similar trend between classical-monocyte frequency and HCV-decline at 4 weeks of therapy in HCV mono- infected subjects (r= -0.250, p=0.180, not shown). Baseline sCD14 negatively correlated with HCV-decline (Fig. 3.2b), as we have previously reported (148). CD4EM baseline frequency (Fig. 3.2c) and absolute count (not shown) positively correlated with greater

HCV-decline (r=0.300, p=0.016; r=0.273, p=0.036) and negatively correlated with sCD14 levels at baseline (r= -0.254, p=0.048, Fig. 3.2d). In addition, there was a negative correlation between IL-6 level and CD4EM T-cell frequency (r= -0.301, p=0.018 not shown), indicating soluble immune-activation markers negatively associate with CD4 EM frequency. The negative association found between CD4CM and EM frequencies and HCV prior to therapy (Fig 3.1b), was upheld at week 4 of therapy (not shown).

We next examined whether anti-viral effector T-cells play a role in these relationships.

We evaluated CEF-, CMV- and HCV-specific T-cell frequency by IFNγ ELISPOT. Of

HIV-HCV co-infected participants, 52% of PBMC samples of responded to HCV- peptides, while 82% and 84% responded to CEF and CMV peptide pools, respectively.

There was no association between cumulative frequency of HCV-specific T-cell responses and HCV-level or therapy-response. However, previous work has indicated

92 that the response to therapy may be more related to the breadth of the response to HCV than magnitude of response (80, 195, 209, 210). When the number of HCV-peptide pools targeted was examined, we observed a modest trend for enhanced IFNα-dependent HCV- decline among subjects who responded to 5 or more peptide pools (5 was the median number of pools targeted) (p=0.200). Given that CEF-peptide responses are CD8 T-cell- driven, we evaluated potential associations between CD8 T-cells and CMV- or CEF- specific IFNγ-responses. Expectedly, we observed a positive trend between CD8CM and

EM T-cell frequency and CEF-specific IFNγ-response (r=0.258, p=0.080; r=0.215, p=0.150). sCD14 negatively associated with CEF-specific T-cell responses (r= -0.361, p=0.011, Fig. 3.2e). We also found CD4EM T-cell frequency and count positively associated with CEF-specific IFNγ-response (r=0.351, p=0.016; r=0.302, p=0.040), the former shown in Fig. 3.2f. These results support the role of CD4EM T-cells in anti-viral specific immune-function and suggest that sCD14 may be a negative predictor of anti- viral immune function.

Figure 3.2. sCD14 is negatively associated with HCV-decline and CEF-IFNγ response, while CD4EM frequency is positively associated with HCV-decline and CEF-IFNγ response. A-C) Week 0 classical monocyte (CD14brightCD16-) frequency (A), sCD14 (B) and CD4 EM T cell frequency (C) in relation to HCV decline at week 4 IFN-based therapy D) Week 0 plasma sCD14 (ng/mL) in relation to week 0 CD4 EM (CD27-45RO+) frequency (%) E-F) Cytomegalovirus/Epstein-Barr/Influenza (CEF) and Cytomegalovirus (CMV) peptides (National Institutes of Health AIDS Research and Reference Reagent Program, Division of AIDS, NIAID) were assayed at a final concentration of 1μg/mL and 2μg/mL, respectively. Week 0 sCD14 (E) and CD4 EM T cell frequency (F) of HCV+HIV+ co- infected subjects, in relation to CEF-specific IFNγ response (measured as spot-forming units, SFU).

Discussion

T-cell-mediated immunity plays a vital role in HCV-clearance (70, 80, 196), presumably by cytolysis of infected cells and IFNγ-secretion (70, 196, 197). Consistent with prior studies, our data show a negative relationship between both CD4CM and EM T-cell frequencies and HCV-level during HCV-HIV co-infection (Fig. 3.1b). There were similar negative associations between CD8CM and EM T-cell frequencies and HCV- level before start of HCV-therapy. We observed a positive association between CD4EM frequency and CEF-specific T-cell response (Fig. 3.2f), and a trend towards an association between HCV-specific T-cell breadth and IFNα-induced HCV-therapy response, supporting a role for CD4-memory T-cells in anti-viral immune-function.

Furthermore, there was a positive relationship between CD4EM T-cell frequencies and greater IFNα-induced HCV-decline magnitude (Fig. 3.2d). An alternative to CD4 T-cells directly playing a role in viral-control would be that HCV directly or indirectly has a negative effect on CD4CM and EM numbers. To address the effect of HIV, we evaluated these cells during HCV+ mono-infection, where we observed heightened CD4CM and

EM frequencies compared to those in uninfected participants (Fig 3.1a-b), and no relation between CD4CM or EM frequency and HCV-level. The latter suggests that the relationship may be restricted to persons with HIV-infection, possibly uncovering an interaction only when immune function is impaired, as is the case when CD4 numbers and function are reduced during HIV co-infection.

CD4CM and EM T-cell frequencies negatively associated with sCD14 (Fig. 3.2b) and

IL-6, which in turn negatively predict HCV-therapy response (148, 151, 152). These

95 associations would be consistent with a negative impact of these factors on T-cell function. In fact, IL-6 has been shown to impair CD4 T-cell induction of pro-survival factor BCL-2 upon IL-7-stimulation (178), and there is at least one report that sCD14 can directly inhibit CD4 T-cell IL-2-production (201).

For the first time, we show that classical-monocyte frequency is negatively associated with both CD4CM and EM frequencies (Fig. 3.1c) and HCV-decline magnitude at week

4 of PegIFNα/rbv-therapy (Fig. 3.2a), consistent with a potential negative role for classical-monocytes in IFNα-dependent T-cell-mediated HCV-clearance. No association was seen between patrolling- or intermediate-monocytes and HCV-levels, suggesting specificity of the finding. Circulating monocytes have an overlapping phenotype with

Kuppfer cells, the resident liver-macrophages, both characterized by expression of CD14

(211). Circulating monocytes are also capable of infiltrating into the liver and serving as precursors to Kuppfer cells (211). Upon stimulation, classical-monocytes shed CD14

(191), CD163, and produce IP-10, IL-6 and TNF (192, 194). Here sCD14 negatively associated with CEF-specific IFNγ-response (Fig. 3.2e). Together, these data suggest negative roles for classical-monocytes and sCD14 in anti-viral immune-function.

Our results extend our understanding of endogenous and exogenous mechanisms of

IFNα-action and immune-mechanisms of HCV-control. In the IFN-free HCV-treatment era, the majority of treated subjects will achieve sustained virologic response (SVR)

(212). However, a treatment-resistant population exists (212), and HCV- eradication

96 worldwide will likely require a preventative vaccine. Further evaluation of the role of T- cell and monocyte immune-function will inform refinement of therapeutic-strategies towards further improved viral-control and vaccine designs.

Chapter 4: Summary and Significance of Findings

Summary of Findings

Genetic evidence supports that natural killer (NK) cells play a role in control of HCV infection in vivo (68). In vitro, NK cells directly mediate control of HCV, via cytokine secretion and cytotoxicity (89, 116). IL-7 is a homeostatic cytokine with a role in T-cell development, activation, proliferation and cytokine secretion (124, 125). The IL-7 receptor α chain (CD127) is expressed on NK cells, with greatest abundance on the

CD56bright NK cell subset (136). We measured CD127 expression on CD56bright, CD56dim or CD56- NK cell subsets of 25 uninfected, 34 chronic HCV infected treatment-naïve, 25

HIV infected, virally suppressed on antiretroviral therapy (ART) and 42 HCV-HIV co- infected on ART subjects. We observed CD127 expression on CD56brightCD16dim/- NK cell was negatively correlated with HCV viral-loads in HCV mono-infection and HCV-

HIV co-infection. We demonstrate that IL-7 enhances CD56bright NK cell Bcl-2 expression and induces cell cycling, which is impaired during HCV and HIV infection.

IL-7 mediates induction and enhancement of IFNα-induced, CD56bright NK cell CD69 expression, and this is impaired in HIV-infection. IL-7 induces NK cell IFNγ production similarly in samples from control, HCV, HIV and HCV-HIV infected subjects, and augments IFNα-mediated NK cell IFNγ release. While IL-7 induced CD56bright NK cell degranulation appeared intact within all groups, we observed impaired IL-7-mediated NK cell cytolytic function within HCV and HIV infection. We have demonstrated that IL-7- induced pSTAT5 signaling is impaired in NK cells of subjects with chronic viral- infection, which is reversible upon 6 months of IFN-free HCV therapy. These results implicate IL-7-dependent NK cell activation and effector function as contributing to control of chronic viral-infection.

NK cells are known to interact with other innate immune cells, including dendritic cells and monocytes, via bi-directional cross talk (89, 97, 114, 117, 139, 213). These reciprocal interactions are responsible for regulation of each immune cell’s activities, which ultimately shapes the adaptive immune response. NK cells are activated by a multitude of

Th1-associated cytokines, IL-12, IL-15, IL-18 and IL-21, produced by both DCs and monocytes (97, 115, 117). NK cells in turn mediate DC and monocyte maturation, promoting effective T cell priming (106, 114, 138). However, DCs and monocytes are also capable of negatively regulating NK cell activation and effector function. Production of both immunosuppressive cytokines, such as IL-10, and inflammatory mediators can perturb anti-viral NK cell activity (89, 116, 166). Monocytes are potent producers of IL-6 and sCD14 (146, 164, 193), which both have been shown to inhibit IL-7-mediated activities and T-cell function (178). Here, we have observed trends towards a positive relationship between classic CD14brightCD16- monocytes and IL-6 and sCD14. Previous work has shown a positive relationship between plasma sCD14 levels and CD56-16+ NK cell frequency within HCV infection (148). Our studies have demonstrated a negative relationship between both sCD14 and CD56-16+ NK cell frequency and response to

HCV therapy (149), indicating a potential role for sCD14 and this dysregulated NK cell subset in control of HCV.

Soluble immune-activation markers, produced in part by CD14brightCD16—monocytes, and monocyte activation have been shown to predict disease-progression and poor IFNα- treatment response (148, 190). We evaluated relationships among immune-activation,

100 monocyte phenotype, CD4-memory T-cells and HCV-, CMV- and CMV/EBV/Influenza

(CEF)-specific IFNγ-response, before and during IFNα-treatment. Effector-memory and central-memory CD4-T-cell frequencies were lower in HCV+HIV+ than uninfected- donors, and correlated negatively with HCV-level, CD14brightCD16--monocytes and plasma sCD14. sCD14 and CD14brightCD16- monocytes negatively correlated with IFNα- dependent HCV-decline. sCD14 negatively associated and CD4 effector-memory T-cells positively-associated with CEF-specific IFNγ-response. These data support a role for memory-CD4 T-cells in HCV-containment, and link immune-activation and

CD14brightCD16--monocyte frequency to failure of interferon-dependent HCV-clearance.

While the full effect of immune activation and monocyte interactions on NK cell phenotype and function are unclear, there are potential associations to be found. In fact, immune activation prior to vaccination has been negatively associated with immune protection (214). Before vaccination, NK cell activation and exhaustion, as well as a proinflammatory monocytes, were associated with reduced CD8 T-cell and B-cell responses to yellow fever (214). The potential implications of this data could connect the effect of immune activation on NK cell anti-viral function and ability to polarize an effective adaptive immune response. Further studies are warranted to understand the effect of monocyte-derived soluble mediators of immune activation on NK cell function, which ultimately affects the adaptive immune response.

101

Chapter 5: Future Directions Determine the role of soluble immune activation mediators on observed IL-7-mediated

NK cell function and anti-viral CD4 T-cell response

102

Characterizing immune activation during HCV therapy

Plasma IL-6 and sCD14 are known to be heightened within HCV and HIV infection, and contribute to a state of chronic immune activation (147, 149, 151, 178, 193).

Additionally, ENPP2 (autotaxin, ATX), an enzyme that leads to production of lysophosphatidic acid (LPA) from lysophosphatidylcholine, has been shown to be over- expressed in HCV infected patients with human hepatocellular carcinoma, and correlates with inflammation and liver cirrhosis during HCV infection (215). We have also recently demonstrated that ENPP2 is heightened within HCV-HIV co-infected subjects, which tended to associated with APRI, indicating potential implications in liver inflammation.

Similarly, forms of LPA appear to be elevated within HIV mono-infection (216). Here, we measured these soluble factors prior to the start of HCV-therapy. Compared to uninfected donors, we saw enhanced levels of IL-6 and sCD14 in HCV mono-infected subjects (Figure 4.1), as well as ENPP2, consistent with previous findings (147, 216).

103

A B

Figure 4.1: Plasma IL-6 and sCD14 are elevated in HCV mono-infection. A-C. Plasma IL-6 (A) and sCD14 (B) were measured by ELISA of uninfected donors (UD, n=25), HCV mono-infected subjects (n=34), HCV-HIV co-infected subjects (n=64) and HIV mono-infected subjects (n=25, IL-6 only). Judge, CJ, unpublished.

Further elucidate the role of soluble immune activation mediators on CD4 memory T-cell

activity

There have been many similar observations of elevated soluble immune activation

mediators within HCV and HIV infections, including IL-6 and sCD14 (147-153, 216),

although the effects of these soluble factors on the anti-viral immune response are not

entirely known. Our data has indicated a negative role for soluble mediators of immune

activation, including sCD14 and IL-6, on CD4 memory T-cell frequency and anti-viral

immune function (149). However, it is unclear if these negative associations observed

reflect a direct role for these inflammatory mediators on CD4 memory T-cell activity.

Previous findings have shown that IL-6 inhibits CD4 T cell response to IL-7 (178). IL-6

104 has also been shown to skew the CD4 T-cell response towards a Th2 phenotype rather than the anti-viral Th1 phenotype via autocrine production of IL-4 (217). sCD14 has been demonstrated to inhibit CD4 T-cell IL-2 and IFNγ production, as well as antigen- mediated PBMC proliferative response (201). We have shown that LPA, the enzymatic product of ENPP2, enhances CD4 T-cell CD38 and HLA-DR co-expression, cellular markers of immune activation (147). Together, these ex vivo and in vitro findings offer a potential direct negative effect of these soluble factors on anti-viral CD4 T cell function.

To further investigate this negative effect of these soluble mediators anti-viral immune function, IFNγ release by ELISPOT assay should be measured upon treatment of CD4 T- cells with or without LPA, IL-6 and/or sCD14 upon exposure to HCV, CMV or CEF peptides, expecting to see impaired anti-viral responses. This work is important to better understand the negative association between immune activation mediators and CD4 T- cell frequency and immune function, vital in establishment and maintenance to an effective anti-viral response.

105

Determine the effect of soluble immune activation mediators on IL-7-mediated NK cell activity

Based on published works in HIV, which have shown a negative correlation between T cell CD127 expression and IL-6 (56), we expected to observe a similar negative association between CD56bright NK cell CD127 expression and IL-6 levels, which are elevated in HCV infection (Figure 4.2, 55). Previous in vitro studies have additionally shown that IL-6 treatment of T-cells leads to reduced CD127 expression and IL-7 induced pro-survival markers (56,75). Within uninfected donors, we found a negative association between plasma IL-6 levels and CD56bright NK cell CD127 expression (r= -

0.557, p=0.009). However, we found that plasma IL-6, which is heightened within chronic HCV mono-infection (Figure 4.1a), is positively associated with CD56bright NK cell CD127 expression within HCV mono-infected subjects (r=0.4000, p=0.030). A plausible explanation for the positive association seen exclusively in the HCV mono- infected group is that both CD56bright NK cells and IL-6 are associated with inflammation in chronic viral infection. IL-6 is a key component of the inflammatory response (55, 82) and CD56bright NK cells express homing receptors that direct them to sites of inflammation and secondary lymphoid sites, such as CXCR3, CCR2, CCR5, and CCR7

(67, 83). Therefore, this positive relationship seen between IL-6 and CD56bright NK

CD127 expression may indicate that both are involved at sites of inflammation within

HCV infection, rather than IL-6 directly increases CD56bright NK CD127 expression.

Another potential reason for this displayed negative correlation is related to liver damage.

With severe chronic HCV, there is increased liver damage and cirrhosis, which

106 eventually leads to less liver tissue and subsequently reduced liver inflammation and

HCV replication, contributing to loss of HCV reservoir (81). To account for liver damage, an APRI score is used, a ratio of AST:platelet levels. Increased APRI values indicates liver damage and possible progression to cirrhosis (81). Recent works have shown that IL-6 levels are heightened in HCV subjects with higher indices of APRI (55).

In this current data set, IL-6 negatively correlated with albumin (p = 0.018, not shown), also indicative of liver dysfunction (81). These soluble marker parameters indicative of liver damage are by no means ideal, and dissecting whether high APRI is reflective of a combination of liver inflammation and damage, or damage itself can make for difficulty in interpreting relationships between a number of soluble inflammation markers and liver damage-state. To account for this, obtaining liver biopsy and fibroscan in parallel will allow better determination of state of liver inflammation and damage.

However, we will determine involvement of IL-6 on CD127-mediated HCV directed NK effector functions via in vitro assays. We expect that IL-6 will contribute to NK polarization toward cytotoxicity, as IL-6 has been directly implicated in enhanced NK activation and cytotoxic activity, although it is unable to induce IFNγ production (32, 57

58). While LPA reportedly inhibits NK cytotoxic function (62), how it affects NK IFNγ secretion is unclear. We anticipate therefore that the ratio of cytotoxicity to IFNγ secretion will be higher upon IL-6 or LPA treatment than seen with IL-7 treatment, indicative of immune activation-mediated inflammation, as described in Figure 1.1.

While the data may strongly suggest that IL-6 and LPA augment CD127-mediated NK effector function, it will not directly show which NK cell subset(s) are most affected. To

107 determine this, we will flow sort bead purified NK cells by expression of CD3, CD56, and CD16 to further analyze CD56neg, CD56dim and CD56bright NK cell subsets.

Another potential explanation for this unexpected finding is that increasing IL-6 is reflective of a key cell type in HCV clearance. Monocytes and dendritic cells, which both carry out cross-talk functions with NK cells and are vital to NK activation and function, can secrete IL-6 (63). Finally, these associations between IL-6 and HCV load, and IL-6 and CD56bright NK cell CD127 expression do not demonstrate direct cause and effect. Future studies are necessary to determine how the immune response to HCV affects IL-6 production, and how this in turn alters NK cell effector function within chronic viral infection.

Elucidate the effect of removal of HCV on IL-7-mediated NK activity and CD4 memory

T-cell frequency and function after DAA therapy

Due to the numerous impairments observed in IL-7-mediated NK cell activity in both

HCV and HIV infections, as well as the striking negative associations seen among CD4 memory T-cell subsets and HCV level, we inquired if there was a potential direct inhibitory role of either virus. However, as previously indicated, soluble immune mediators may also be playing a major role in both IL-7-mediated NK cell activity, as well as in CD4 memory T-cell frequency and anti-viral function. Given that we and others have demonstrated that soluble immune mediators are elevated within chronic viral infection (Figure 4.1), HCV and HIV may be indirectly impairing IL-7 signaling

108 due this state of immune activation. We have previously shown that sCD14 increases during IFN-containing therapy (148), and we recapitulate that finding along with increased levels of IL-6 (Figure 4.2), while these soluble factors, in addition to ENPP2, begin to normalize on IFN-free therapies (216).

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A B

Figure 4.2.: Plasma sCD14 and IL-6 levels are increased during IFN-containing HCV therapy. A-B. Plasma sCD14 (A) and IL-6 (B) levels were measured by ELISA of HCV-HIV co-infected subjects (n=64) at the start of IFN-containing HCV therapy (week 0), and at weeks 4 and 12. Judge, CJ, unpublished.

Dissect the effects of HCV and immune activation on IL-7-mediated NK cell activity

Our findings in Figure2.6d support that removal of HCV with IFN-free DAA therapy

restores IL-7-mediated CD56bright NK cell phosphorylation of STAT-5. This indicates a

potential inhibitory role for chronic HCV infection on IL-7-mediated NK cell activity.

However, whether this is a direct effect of HCV, or reflects the effects of reduction of

immune activation on IL-7-mediated NK cell activity remains to be determined.

To further dissect if the effect of HCV is potentially mediating an indirect effect via

elevation of sCD14 and IL-6, we can compare IL-7-mediated NK activity of HCV-

cleared subjects on IFN-containing therapy with those who cleared HCV on IFN-free

regimens. During IFN-containing HCV therapy, sCD14 and IL-6 increase, while both of

110 these markers of immune activation decrease on IFN-free therapy. By comparing these two therapy types, we aim to understand how these soluble factors potentially impact this innate cell signaling pathway. Alternatively, both groups achieving SVR by IFN- containing and IFN-free therapies may ameliorate IL-7 NK cell signaling activity, which was observed to be impaired in HCV-infected subjects, indicating that HCV is directly impairing this function.

Understanding the role of HCV and soluble immune activation mediators on CD4 memory T-cell frequency and anti-viral response

In Figure 3.1a-b, we demonstrated reduced frequency of CD4 memory T-cells within

HCV-HIV infection, while not observed in HCV mono-infection. To further determine any potential role of HCV, we can investigate the potential changes in CD4 effector and central memory T-cells after removal of HCV. We speculate that these observed reductions in CD4 effector and central memory T-cells within HCV-HIV co-infection reflect an effect of HIV, based on heightened frequencies of both CD4 effector and central memory T-cell frequencies within HCV mono-infection (Figure 3.1a-b).

There is evidence for a strong role of CD4 memory T-cells in clearance of HCV via IFNγ release (71, 78-80, 134, 195). Our data supported this evidence, with positive associations found among CD4 effector memory T-cells and HCV-decline on therapy and CEF-IFNγ response (Figures 3.2c, 3.2f, respectively). Meanwhile, we observed negative associations between both IL-6 and sCD14 and CD4 effector memory T-cells and HCV-

111 decline on therapy. We also demonstrated a negative correlation between sCD14 levels and CEF-IFNγ response, suggesting a potential negative role for soluble factors of immune activation and anti-viral immune function. Future studies are needed to determine if pre-exposure to soluble factors such as IL-6 and sCD14 dampen the CD4 memory T-cell response.

Understand the effect of monocytes on IL-7-mediated NK cell activity and CD4 T- cell anti-viral function

Monocytes are also known contributors to production of soluble mediators of immune activation, including sCD14, IP-10, IL-6, TNFα and IL-1β (146, 164, 193), which are elevated in HCV and HIV infections (147-153). Similarly, we have also recently shown that ENPP2 positively associates with sCD14, IL-6 and sCD163 within HCV mono- infection (216), indicating a potential indirect link between ENPP2 and its enzymatic product LPA and monocyte activation. Here, we observed positive associations of classical monocyte frequency and the dysregulated CD56- NK cell subset within HCV-

HIV co-infection (Figure 4.3a), as well as a negative association between the indirect monocyte activation marker sCD14 and CD56bright NK cell frequency (Figure 4.3b) within HCV mono-infection.

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A B

Figure 4.3: Classical monocytes positively correlate with CD56-16+ NK cells, while sCD14 negatively associates with CD56bright NK cells in HCV infection. A-B. CD56-16+ (A) and CD56bright16dim/- (B) NK cell frequencies, measured as proportion of total NK cells of PBMC, were positively correlated with classical monocytes, defined as CD14brightCD16- (A) and negatively correlated with plasma sCD14 (B). Judge, CJ, unpublished.

The effect monocytes and/or monocyte-derived soluble factors on NK cell phenotype and

function is not fully understood. However, literature indicates that immune activation

perturbs NK cell anti-viral function, further polarizing NK cells in chronic HCV towards

non-specific cytotoxicity as well as reduced IFNγ production (92, 122). It is of interest to

understand how chronic viral infection affects monocyte cytokine and soluble mediator

production, which can in turn determine interactions with NK cells. Monocytes are

capable of releasing IL-15 and inflammasome-dependent IL-18, which directs NK cells

to produce IFNγ (198). However, in chronic viral infection, monocytes also produce

moderate to high levels of IL-10 and TGF-β (218, 219). In fact, it has been shown that

NS5A of HCV directly interacts with monocytes, leading to increased release of IL-10

and TGF-β (220). In turn, TGF-β has been demonstrated to downregulate NK activating

receptor NKG2D, which impairs NK cell IFNγ release and degranulation (220).

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Furthermore, IL-10 induction leads to increased upregulation of PDL-1 expression on monocytes within HCV and HIV infections, as well as documented induction of PD-1 on

CD4 T-cells (219, 221-223). CD56bright NK cells have also been demonstrated to upregulate PD-1 expression within chronic HCV infection (223). While this is in part a mechanism to reduce inflammation, there is subsequent suppression of NK cell IFNγ release and cytolytic function (218), as well as CD4 T-cell exhaustion and inhibition of

CD4 T-cell anti-viral function (218, 219). This exhaustive phenotype on both NK cells and CD4 T-cells has been negatively associated with sustained-virologic response (SVR) within HCV-infected subjects (223). More studies are necessary to demonstrate if classical monocytes impair CD4 T-cell anti-viral immune function and IL-7 mediated NK cell activity via interaction of PDL-1 on monocytes with PD-1 on NK cells and CD4 T- cells and production of soluble immune activation mediators.

Does IL-7 promote NK-dependent T-cell priming?

Determine the potential role of IL-7 on NK editing of DCs, monocytes and macrophages

DC and NK cell bi-directional cross-talk dictates the ultimate function of each cell type.

In the lymph node, where HIV replication occurs, the NK cells present are typically regarded as immunoregulatory yet immature CD56bright, expressing little to no KIRs and high surface NKG2A (89, 111, 123). NK cells are enriched in the liver, making up between 30-50% of all lymphocytes (89, 218, 224), and have a much greater proportion of CD56bright NK cells than found in the peripheral blood, composing up to approximately

50% of all hepatic NK cells (225-227). NK cells of the liver are less cytolytic than their peripheral blood counterpart and lack CD16 expression (89, 111), and are characterized

114 by their ability to produce polarizing cytokines such as IFNγ and TNFα (89). However there is still a paucity of literature on hepatic-derived NK cells and more studies are required to further understand the phenotype and function of these NK cells.

Upon activation and/or exposure to antigen, activated DCs promote NK cell effector function via release of an array of cytokines, including IL-12, IL-15 (228, 229), as well as

IL-7 (230). We have observed IL-7 nearly exclusively activates the CD56bright NK cell subset (Figure 2.2a). Our data also demonstrated that IL-7 mediates NK cell IFNγ release (Figure 2.3a-c), confirmed to act on the CD56bright NK cell subset. Interestingly, we observed that IL-7-mediated NK cell IFNγ release was the only IL-7-enhanced NK cell effector function that remained intact in HCV, HIV and HCV-HIV infected subjects

(Figure 2.3c). Potentially, DC-derived IL-7 might be promoting NK cell activation and

IFNγ release within the lymph node and liver, which could aide in control of HIV and

HCV, respectively. Further studies examining the effect of IL-7 on NK cells of the lymph node and liver are needed, with the aim to demonstrate that this IL-7-enhanced NK cell

IFNγ release is remains intact in the lymph node and liver of HCV and HIV infected subjects.

NK cells are vital in the maturation process of APCs, including DCs, monocytes and macrophages. It is therefore of interest to elucidate the potential effect of IL-7 on NK cell-dependent APC maturation. APC maturation is measured by upregulation of co- stimulatory molecules CD80, CD86, as well as HLA-DR and CD83 (96, 140). After maturation, monocytes, macrophages and DCs become potent in T-cell priming (218).

We and others have demonstrated that upon activation, NK cells are more effective at

115 promoting DC maturation (96, 140, 218). While we saw no effect of IL-7 on NKp30 expression, which is positively involved in NK editing of DCs as well as monocyte and macrophage maturation (213), it is possible that IL-7 may promote upregulation of other

NCRs that are involved, such as NKp44 and NKp46. In contrast, IL-7 may also be leading to reduction of inhibitory NKRs, although we saw no effect of IL-7 on NK cell

NKG2A expression. NK release of TNFα and IFNγ also promotes APC maturation (140,

141), which was observed to be enhanced upon stimulation with IL-7. Although unobserved here, it has been shown that monocytes exposed to IFNγ have subsequent prolonged TNFα release as well as reduced IL-10 release (231), which inhibits NK cell- mediated APC maturation (213, 232). IL-7 may promote NK cell editing of APC maturation via favoring an anti-viral phenotype by production and release of IFNγ and

TNFα and potential subsequent inhibition of 1L-10.

Potential of improving Th1 polarization with IL-7-exposed NK cells

Enhancing APC maturation will ultimately promote effective anti-viral T-cell priming and polarization. To elucidate the effect of IL-7-mediated NK-dependent APC maturation on the ability to polarize a Th1 response, we can evaluate differences between T-cells cultured with either monocytes and DCs matured with NK cells treated with or without

IL-7. If indeed IL-7 enhances NK cell ability to mature APCs, we expect that T-cells cultured with DCs or monocytes that have undergone maturation in presence of IL-7- treated NK cells to release enhanced amounts of IFNγ and TNFα, characteristic of a Th1 response (89, 127, 128).

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While the ability of NK cells to mature monocytes and DCs into effective APCs is a crucial indirect role in shaping the anti-viral T-cell response, NK cells can also directly determine the T-cell response. NK cells are able to polarize CD4 T-cells to a Th1-like response by producing anti-viral cytokines IFNγ and TNFα (89, 111). Our data indicates that IL-7 pre-exposure promotes this anti-viral cytokine release (Figure 2.3), and potentially suggests that CD4 T-cells cultured with IL-7-treated NK cells or IL-7-induced

NK cell cytokine release will promote a Th1 response, required for HCV clearance.

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Concluding Remarks

We have demonstrated that CD56bright NK cell CD127 expression negatively associates with plasma HCV level in both HCV mono-infection and HCV-HIV co-infection.

Investigation of this relationship at a functional level indicates IL-7 promotes CD56bright

NK cell activation, cell-cycling, IFNγ release and killer function. During chronic HCV and HIV infections, there are overlapping, yet distinct impairments in IL-7-mediated NK cell activity. Specifically, during both HCV and HIV infection, IL-7-mediatied NK cell cycling, cytolytic activity, and STAT-5 phosphorylation were impaired. Additionally, IL-

7-mediated CD56bright Bcl-2 induction was impaired in HCV infection, while IL-7- enhanced NK cell granzyme B release and CD56bright NK CD69 upregulation was impaired in HIV infection. These data implicate IL-7-dependent NK cell function in control of HCV infection and present another potential immune evasion mechanism of

HCV and HIV.

Whether these impairments in IL-7-dependent NK cell function are direct effects of HCV and HIV virus, or indirect effects related to long-standing infection remains to be shown.

HCV infection may indirectly contribute to lower IL-7 levels, because of liver damage and reduced IL-7 synthetic function. However, we also speculate that the impaired IL-7- mediated activities may be attributable to soluble factors of immune activation, including sCD14 and IL-6, which are elevated in chronic HCV and HIV infections. This may affect immune cell phenotype and function. In fact, we observed a negative association between

CD56bright NK cell frequency and plasma sCD14 levels within HCV mono-infected

118 subjects. In both HCV and HIV infections, there are increased numbers of the dysregulated CD56neg NK cells, which are impaired in cytolytic activity and anti-viral cytokine production (121). Within HCV-HIV co-infected subjects, we found a positive correlation between CD56neg NK cell numbers and classical monocyte numbers, the same cells that produce significant amounts of sCD14 and IL-6 (146, 164). Overall, these findings support a role for NK cells in control of HCV infection, provide a link between

IL-7-dependent NK cell function and control of HCV during HCV and HIV infection, and indicate a connection between monocytes and NK cell subset frequency. More studies are required to better elucidate the role of immune activation and monocyte-NK cell interactions on IL-7-mediated NK cell activity.

We also observed negative roles of classical monocytes and immune activation on other crucial mediators of HCV clearance, CD4 memory T-cells. Within HCV-HIV co-infected subjects, classical monocytes, sCD14, and IL-6 negatively associated with CD4 effector and central memory T-cells, which both were negatively associated with HCV level in

HCV-treatment-naïve subjects. CD4 effector memory T-cells were positively associated with anti-viral immune function as measured by CEF-IFNγ response, as well as HCV decline on HCV-therapy. In contrast, classical monocytes and sCD14 negatively associated with HCV decline, and sCD14 also negatively correlated with CEF-IFNγ response, suggesting classical monocytes and soluble factors of immune activation have a negative role in anti-viral immune phenotype and function. These data indicate a need for the further study of the interplay between monocytes, immune activation, the innate immune response and control of HCV.

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