Role of plasmacytoid dendritic cells and other accessory ... · Role of plasmacytoid dendritic cells and other accessory cells in the activation of human natural killer cells by herpes

Role of plasmacytoid dendritic cells and other accessory cells in the

activation of human natural killer cells by herpes simplex virus type 1

Die Rolle plasmazytoider dendritischer Zellen und anderer akzessorischer

Zellen in der Aktivierung humaner natürlicher Killer-Zellen durch

Herpes-Simplex-Virus-1

Der Naturwissenschaftlichen Fakultät

der Friedrich-Alexander-Universität

Erlangen-Nürnberg

zur

Erlangung des Doktorgrades Dr. rer. nat.

vorgelegt von

Karin Petra Vogel

aus Nürnberg

Als Dissertation genehmigt von der Naturwissenschaftlichen Fakultät

der Friedrich-Alexander-Universität Erlangen-Nürnberg

Tag der mündlichen Prüfung 23.01.2015

Vorsitzender des Promotionsorgans Prof. Dr. Jörn Wilms

Gutachter Prof. Dr. Barbara Schmidt

Prof. Dr. Andreas Burkovski

1

Table of contents

1 Summary .......................................................................................................................... 3

1 Zusammenfassung ........................................................................................................... 4

2 Introduction ..................................................................................................................... 5

2.1 Herpes simplex virus type 1 ................................................................................ 5

2.2 Natural killer cells ............................................................................................... 7

2.3 Plasmacytoid dendritic cells .............................................................................. 10

2.4 Mononuclear phagocytes ................................................................................... 12

2.5 Interactions of PDC and NK cells in HSV infection ......................................... 15

3 Rationale ........................................................................................................................ 16

4 Materials and Methods ................................................................................................. 17

4.1 Materials ............................................................................................................ 17

4.1.1 Instruments .......................................................................................... 17

4.1.2 Consumables ........................................................................................ 18

4.1.3 Reagents .............................................................................................. 19

4.1.4 Software .............................................................................................. 20

4.1.5 Commercial Kits .................................................................................. 20

4.1.6 Cell Culture ......................................................................................... 20

4.1.7 Viruses ................................................................................................. 21

4.1.8 Media and Buffers ............................................................................... 21

4.1.9 Antibodies ............................................................................................ 22

4.1.10 Isotype Controls ................................................................................... 24

4.2 Methods ............................................................................................................. 25

4.2.1 Isolation of primary human cells ......................................................... 25

4.2.2 Determination of cell numbers ............................................................ 26

4.2.3 Herpes simplex virus type 1 stocks ..................................................... 27

4.2.4 PDC supernatants ................................................................................ 28

4.2.5 Stimulation and infection of cells with HSV-1 ................................... 29

4.2.6 FACS analysis of cells ......................................................................... 30

4.2.7 Determination of secreted cytokines within supernatants ................... 31

4.2.8 Quantification of HSV-1 DNA ............................................................ 34

2

4.2.9 Virological analysis of hyperproliferative lesions ............................... 34

4.2.10 Statistical analysis ............................................................................... 34

5 Results ............................................................................................................................ 35

5.1 Stimulation of PBMC with HSV-1 leads to NK cell activation ........................ 35

5.2 Only infectious HSV-1 induces NK cell effector functions .............................. 38

5.3 HSV-1 activates NK cells in part via type I IFN induction .............................. 40

5.4 TNF- plays a major role in HSV-1-induced NK cell activation ..................... 43

5.5 Monocytes contribute to HSV-1-induced TNF- production ........................... 47

5.6 Monocytes can be infected by HSV-1 ............................................................... 49

5.7 Monocytes up-regulate MHC-I upon exposure to infectious HSV-1 ............... 51

5.8 HSVd106S affects monocytes similar to HSVGFP ................................................ 56

5.9 Monocytes mediate NK cell effector functions upon HSV-1 infection within the

PBMC context ............................................................................................................. 59

5.10 PDC serve as crucial accessory cell population in NK cell activation by HSV-1-

infected HFF ................................................................................................................ 61

5.11 PDC supernatants inhibit HSV-1 replication in HFF ........................................ 64

5.12 PDC-NK cell interactions are hampered in an HIV-1-infected woman suffering

from persisting genital ulcers ...................................................................................... 67

6 Discussion ....................................................................................................................... 70

7 Abbreviations ................................................................................................................ 85

8 References ...................................................................................................................... 88

9 Publications .................................................................................................................... 99

Summary

3

1 Summary

Herpes simplex virus type 1 (HSV-1), a member of the herpes virus family, is characterized

by a short replication cycle, high cytopathogenicity, and distinct neurotropism. Primary

infection and reactivation may cause severe diseases in immunocompetent and

immunosuppressed individuals. Since studies of human natural killer (NK) cell activation by

HSV-1 are limited, this study investigated mechanisms of NK cell activation by HSV-1 in

vitro, using sucrose gradient-purified UV-inactivated (HSVUV) and infectious (HSVINF)

HSV-1 to stimulate peripheral blood mononuclear cells (PBMC). HSVUV and HSVINF

exhibited distinct stimulatory differences: While both induced IFN- secretion within PBMC

and CD69 up-regulation on NK cells, only HSVINF caused TNF- and IL-1 secretion within

PBMC and NK cell effector functions degranulation and IFN- secretion. IFN- and TNF-

contributed to CD69 up-regulation, and TNF- proved important for IFN- secretion, as

evident from neutralization experiments. Degranulation was independent from IFN-,

TNF-, and IL-1, but dependent on monocytes, as evident from depletion experiments.

Infection experiments evidenced non-productive infection of monocytes by HSV-1,

suggesting recognition of infected monocytes by NK cells as possible cause for degranulation.

MHC-I down-regulation and MICA/MICB expression were excluded as activating signals for

NK cells. Plasmacytoid dendritic cells (PDC), however, proved to suppress HSV-1 replication

within fibroblasts via secreted cytokines. Furthermore, in case of an HIV-1-positive patient

suffering from HSV-2- and HPV-54-induced hyperproliferative lesions, stimulation of PBMC

with HSV-1 resulted in severely reduced IFN- secretion and impaired NK cell activation,

suggesting a role for hampered PDC-NK cell interactions in the patient’s disease. Altogether,

our data suggest a model in which HSV-1-stimulated PDC and monocytes activate NK cells

via IFN- and TNF-, while infection of monocytes induces NK cell effector functions via

TNF--dependent and -independent mechanisms. Furthermore, PDC inhibit HSV-1

replication within susceptible cells and therefore limit viral spread. Thus, PDC and monocytes

appear to have important bystander functions for NK cells to control viral infections.

Zusammenfassung

4

1 Zusammenfassung

Herpes-simplex-Virus-1 (HSV-1), ein Mitglied der Familie der Herpesviren, zeichnet sich

durch einen kurzen Replikationszyklus, hohe Pathogenität, und starken Neurotropismus aus.

Primärinfektion und Reaktivierung können in immunkompetenten und immunsupprimierten

Individuen schwere Krankheiten verursachen. Da es nur wenige Untersuchungen zur

Aktivierung humaner natürlicher Killer (NK)-Zellen durch HSV-1 gibt, wurden in dieser

Arbeit Mechanismen der NK-Zell-Aktivierung durch HSV-1 in vitro untersucht, wofür

mononukleäre Zellen des peripheren Bluts (PBMCs) mit über Succrosegradient

aufgereinigtem UV-inaktivierten (HSVUV) und infektiösen (HSVINF) HSV-1 stimuliert

wurden. HSVUV und HSVINF wiesen deutliche stimulatorische Unterschiede auf: Während

beide zu IFN--Sekretion in PBMCs und CD69-Hochregulierung auf NK-Zellen führten,

induzierte nur HSVINF TNF-- und IL-1-Sekretion in PBMCs sowie die NK-Zell-

Effektorfunktionen Degranulation und IFN--Sekretion. Neutralisationsversuche wiesen die

Beteiligung von IFN- und TNF- an der CD69-Hochregulierung nach, sowie die

Wichtigkeit von TNF- für die IFN--Sekretion. Die Degranulation war nicht abhängig von

IFN-, TNF- oder IL-1, sondern von Monozyten, wie Depletionsversuche zeigten.

Infektionsversuche bewiesen die nicht-produktive Infektion von Monozyten durch HSV-1,

was auf die Erkennung infizierter Monozyten durch NK-Zellen als mögliche Ursache der

Degranulation hindeutet, wobei MHC-I-Herabregulierung und MICA/MICB-Expression als

aktivierende Signale für NK-Zellen ausgeschlossen wurden. Plasmazytoide dendritische

Zellen (PDCs) unterdrückten dagegen die HSV-1-Replikation in Fibroblasten über Zytokin-

Sekretion. Im Fall einer HIV-1-positiven Patientin mit HSV-2- und HPV-54-induzierten

hyperproliferativen Läsionen resultierte die Stimulation von PBMCs mit HSV-1 in stark

verringerter IFN--Sekretion und NK-Zell-Aktivierung, was eine Rolle von verminderten

PDC-NK-Zell-Interaktionen in der Krankheit der Patientin andeutet. Unsere Daten legen ein

Modell nahe, nach dem HSV-1-stimulierte PDCs und Monozyten NK-Zellen über IFN- und

TNF- aktivieren, während die Infektion von Monozyten NK-Zell-Effektorfunktionen über

TNF--abhängige und -unabhängige Mechanismen induziert. PDCs inhibieren außerdem die

HSV-1-Replikation und dadurch eine Ausbreitung des Virus. Die Anwesenheit von PDCs und

Monozyten erscheint daher wichtig für die Kontrolle viraler Infektionen durch NK-Zellen.

Introduction

5

2 Introduction

2.1 Herpes simplex virus type 1

Herpes simplex virus type 1 (HSV-1) belongs to the family of herpesviruses and is highly

prevalent worldwide (Bernard Roizman et al., 2007b). It possesses a linear DNA genome

encoding more than 90 genes, which is enclosed by a capsid built of diverse viral capsid

proteins. The capsid itself is surrounded by the so called tegument, which consists of various

viral tegument proteins. A host cell-derived membrane containing several viral glycoproteins

envelops the viral particle (FIG. 1) (Bernard Roizman et al., 2007e). The viral DNA exists as

circular episome within the nucleus of the infected cell. Viral gene expression is organized

into three phases during replication: immediate-early or , early or and late or (Bernard

Roizman et al., 2007d).

FIG. 1. Herpes simplex virus type 1 (HSV-1) particle. The linear viral DNA genome is enclosed by the capsid

which itself is surrounded by the tegument. Both capsid and tegument are composed of viral proteins. A host

cell-derived membrane containing several viral glycoproteins (gB - gN) envelops the viral particle.

Together with HSV-2 and varicella zoster virus (VZV), HSV-1 belongs to the subfamily of

-herpesviruses and displays high cytopathogenicity, a short replication cycle and a distinct

neurotropism (Philip E.Pellett and Bernard Roizman, 2007). Primary infection and lytic

replication take place at oral or genital mucocutaneous sites. From there, viral particles are

transported along peripheral sensory nerves to the trigeminal or dorsal root ganglia, where

HSV-1 establishes lifelong latency. After reactivation viral particles are transported back to

the primary infection site, where lytic replication leads to viral shedding and potentially but

not necessarily to disease (Bernard Roizman et al., 2007c). Common symptoms of

Introduction

6

reactivation are cold sores and genital herpes. In rare cases, however, reactivation of HSV-1

as well as primary infection can cause severe diseases in immunocompetent individuals, like

acute retinal necrosis (ARN) or encephalitis, while in immunosuppressed individuals it can

lead to disseminated, systemic infections (Bernard Roizman et al., 2007a).

HSV-1 infections are tightly controlled by the immune system, including a wide variety of

immune cells (Cunningham et al., 2006). Cells of both innate and adaptive immunity

participate in the suppression of HSV-1 replication, and interactions between different cell

types take place within and across the innate-adaptive barrier (Schuster et al., 2011). Innate

immunity is crucial for the early, fast response to primary HSV-1 infection (Ashkar and

Rosenthal, 2003), and also appears to play a role in reactivation (Donaghy et al., 2009; Kittan

et al., 2007). Type I interferons (IFN), mainly produced by plasmacytoid dendritic cells

(PDC) (Siegal et al., 1999; Cella et al., 1999), are key factors in the anti-herpesviral response

(Zhang et al., 2007). They lead to an antiviral state of HSV-1 infected and susceptible cells on

the one hand (Härle et al., 2001), and they activate cells of the innate as well as the adaptive

immune system and thus trigger the immune response on the other hand (Gill et al., 2011;

Tough et al., 1996). Natural killer (NK) cells mediate recognition and killing of infected cells

as well as early production of IFN- (Lodoen and Lanier, 2006). Adaptive immunity appears

to contribute to maintenance of latency and limiting of viral spread. While the role of humoral

immunity is not clear, contributions of cell-mediated immunity against HSV-1, especially the

role of cluster of differentiation (CD)4+ T cells and CD8

+ T cells, have been well described

(Johnson et al., 2008; Koelle et al., 1998; Ghiasi et al., 1999).

Introduction

7

2.2 Natural killer cells

NK cells are a large granular lymphocyte subset distinct from B and T cells, which aroused

the interest of researchers due to its ability to lyse tumor cells as well as virus-infected cells

without prior sensitization and without restriction by major histocompatibility (MHC)

antigens (Trinchieri, 1989). Human NK cells, which are defined as CD3-CD56

+ cells, divide

into two phenotypic subsets, according to their expression of CD56 and CD16 (Cooper et al.,

2001a). CD56 was found to be identical with neural cell adhesion molecule (NCAM) (Lanier

et al., 1989), which belongs to the immunoglobulin (Ig) superfamily and mediates homotypic

adhesion between cells. It is expressed in nervous tissues of many vertebrates and plays a

major role in the embryonic development of the nervous system (Rutishauser and Jessell,

1988). CD16 is part of the low affinity fragment, cristallizable receptor IIIA (FcRIIIA),

which recognizes and binds the Fc part of antibodies bound to cell-associated antigens,

thereby inducing antibody-dependent cellular cytotoxicity (ADCC) towards opsonized target

cells (Leibson, 1997). CD56bright

CD16dim/-

cells account for about 10%, CD56dim

CD16bright

cells for about 90% of circulating NK cells. Besides their distinct phenotype researchers

observed functional differences between those two subtypes (FIG. 2). CD56bright

CD16dim/-

cells, which constitutively express the high affinity interleukin 2 (IL-2) receptor (IL-2R),

proliferate in response to low amounts of IL-2 and primarily account for the secretion of

cytokines such as IFN- or tumor necrosis factor (TNF)-, while CD56dim

CD16bright

cells

exhibit high cytotoxicity, mediated either through binding of activating NK cell receptors to

their ligands or through binding of CD16 to opsonized target cells (Cooper et al., 2001a).

Introduction

8

FIG. 2. Natural killer (NK) cell subsets. CD56bright

CD16dim/-

cells show high expression of CD56 and low or no

expression of CD16, they constitutively express the high affinity interleukin (IL)-2 receptor (IL-2R) and

primarily account for the secretion of cytokines such as interferon (IFN)- or tumor necrosis factor (TNF)-.

CD56dim

CD16bright

cells show low expression of CD56 and high expression of CD16 and exhibit high

cytotoxicity.

In contrast to B and T cells, NK cells recognize their target cells independently of antigen-

specific receptors. The activation status of NK cells is determined by a balance of signals

resulting from binding of inhibitory and activating NK cell receptors to their respective

ligands (Lanier, 2005). Inhibitory receptors recognize MHC class I (MHC-I) molecules,

activating receptors recognize stress-induced or virus-derived molecules on a target cell

(Kärre et al., 1986; Bauer et al., 1999). Cytokines secreted by other immune cells further

influence NK cell activation and functions (Nguyen et al., 2002). NK cells play a crucial role

in the immune defense against various pathogens such as viruses, bacteria and parasites. They

contribute to the control of infection by secretion of IFN- and by killing of infected cells

(Lodoen and Lanier, 2006). NK cells kill target cells via two main mechanisms: via granule-

dependent cytotoxicity, where cytotoxic granules containing perforin and granzymes are

released towards the target cell (Kägi et al., 1994; Metkar et al., 2002), and via stimulation of

death receptors on the target cell by TNF-related apoptosis-inducing ligand (TRAIL) (Zamai

et al., 1998), Fas ligand (FasL) (Arase et al., 1995) or TNF- (Paya et al., 1988). In addition

to their effector functions, NK cells exhibit regulatory functions and engagement in reciprocal

Introduction

9

interactions with various cell types, amongst others T cells, macrophages and dendritic cells

(Vivier et al., 2008).

Studies with NK cell-depleted mice could demonstrate the in vivo contribution of NK cells

particularly in the initial control of HSV infections (Habu et al., 1984; Tanigawa et al., 2000),

but there is also evidence for an accessory role of NK cells in adaptive immunity

(Nandakumar et al., 2008), and even HSV-induced NK cell memory has recently been

described (Abdul-Careem et al., 2012). Humans with NK cell deficiencies show increased

susceptibility to herpesviral infection, indicating an important role for NK cells in human

HSV immunity (Jawahar et al., 1996; Dalloul et al., 2004; Orange, 2002). In vitro studies

showed the ability of NK cells to recognize HSV-1-infected cells, leading to secretion of

IFN- and lysis of infected cells. NK cell activation occurred early enough in infection to

reduce spread of virus progeny and therefore limit viral replication in tissue culture

(Fitzgerald et al., 1985; Leibson et al., 1986). A role in NK cell recognition of HSV-infected

cells has been described for MHC-I molecules, which are known to be down-regulated by the

HSV-1 protein infected cell polypeptide (ICP)47 (Hill et al., 1995; Früh et al., 1995): HeLa

cells infected with HSV-1 or transfected with ICP47 down-regulated human leukocyte antigen

(HLA)-C molecules, which was sufficient to mediate NK cell cytotoxicity by NK cell clones

expressing an inhibitory killer cell immunoglobulin-like receptor (KIR) that recognizes

HLA-C (Huard and Früh, 2000). Other groups have shown that expression of HSV-1

immediate early proteins, particularly ICP0, is necessary and sufficient for NK cell

recognition of HSV-1-infected cells (Fitzgerald-Bocarsly et al., 1991; Chisholm et al., 2007).

In addition, cytokines influence NK cell activity, for example IL-15, which seems to be of

importance in the activation of NK cells in the context of peripheral blood mononuclear cells

(PBMC) (Ahmad et al., 2000), and type I IFN, which seem to be involved in activating NK

cells to lyse HSV-1-infected fibroblasts (Feldman et al., 1992). Also, mouse models suggest

roles for the IFN-/ receptor and hence type I IFN (Gill et al., 2011), IL-18 (Reading et al.,

2007), and dendritic cells as important accessory cells (Kassim et al., 2009; Frank et al.,

2012).

Introduction

10

2.3 Plasmacytoid dendritic cells

In 1999 PDC were identified as major producers of type I IFN (Siegal et al., 1999; Cella et al.,

1999). While they are negative for the expression of lineage markers, they express the specific

receptors blood dendritic cell antigen 2 (BDCA-2) and BDCA-4 (FIG. 3) (Dzionek et al.,

2001; Dzionek et al., 2002). PDC play a crucial role in the immune response to viral

infections. Their contributions to the control of viral infection appear to be not only of a direct

manner, but also of an indirect manner by interacting with various immune cells and thus

linking innate and adaptive immunity (Colonna et al., 2004). Stimulation of PDC with HSV-1

leads to secretion of high amounts of IFN-. This stimulation does not require infectivity,

since ultraviolet light (UV)-inactivated HSV-1 is able to stimulate PDC, and HSV-1 does not

replicate in PDC (Schuster et al., 2010; Donaghy et al., 2009). PDC stimulation occurs via

recognition of the viral genome by toll-like receptor 9 (TLR-9) within endosomes (Krug et al.,

2004), which has also been shown for HSV-2 (Lund et al., 2003).

FIG. 3. Plasmacytoid dendritic cell (PDC). PDC are identified by their expression of blood dendritic cell

antigen 2 (BDCA-2) and BDCA-4 and by lacking expression of lineage markers. They recognize HSV-1 via toll-

like receptor 9 (TLR-9) which is expressed in endosomes. Stimulation by HSV-1 leads to secretion of high

amounts of IFN-.

Coordinated regulation of surface receptors upon stimulation indicates various aspects of PDC

function, like attraction to inflamed tissue, antigen recognition and subsequent migration into

secondary lymphatic tissue (Schuster et al., 2010). In fact, upon vaginal HSV-2 infection,

PDC are recruited to the infected tissue and suppress viral replication (Lund et al., 2006).

Introduction

11

Furthermore, virally stimulated PDC are able to induce migration (Megjugorac et al., 2004)

and also activation of cells of the innate and adaptive immune system (Feldman et al., 1992;

Kadowaki et al., 2000). The capacity of PDC to engulf antigen and present it to T cells is

controversially discussed (Villadangos and Young, 2008). The significance of PDC in HSV

infections has been demonstrated in different mouse models. In this respect, Lund et al.

observed an increase in pathogenesis of genital HSV-2 infections after antibody-dependent

PDC depletion (Lund et al., 2006), while Swiecki et al. found that PDC depletion in

CLEC4-DTR mice diminished type I IFN as well as pro-inflammatory cytokine production,

NK cell activation and CD8+ T cell responses during systemic HSV-1 and HSV-2 infections

(Swiecki et al., 2013).

Introduction

12

2.4 Mononuclear phagocytes

Mononuclear phagocytes constitute an important and early component of the immune system.

Monocytes are normally circulating in the blood, while macrophages and dendritic cells,

which represent differentiated stages of monocytes, reside in lymphoid and non-lymphoid

tissues. Macrophages serve as first line defense against invading pathogens as well as

initiators of inflammation, and dendritic cells are particularly important in initiating and

supporting adaptive immune responses. Upon pathogen invasion and inflammation, blood

monocytes support resident macrophages and dendritic cells by infiltrating the tissue and

differentiating into one or the other, depending on the cytokine milieu (Michael Ehrenstein et

al., 2008b; van and Cohn, 1968; Randolph et al., 1999). Mononuclear phagocytes are

equipped with receptors that recognize a wide range of ligands, like pathogen-derived

molecules, so called opsonizing molecules of the humoral immune system that are bound to

pathogens, and chemokines as well as cytokines, which enables them to recognize pathogens

directly and indirectly, and to communicate with other cells of the immune system. Two of

those receptors are predominantly expressed by monocytes and macrophages: CD14 binds

bacterial lipopolysaccharide (LPS), thereby leading to its recognition by TLR-4, and CD64,

also known as FcRI, binds the Fc part of antibodies and thereby recognizes opsonized

pathogens (FIG. 4) (Michael Ehrenstein et al., 2008c; Michael Ehrenstein et al., 2008d). Their

importance in pathogen defense is due to their secretion of various inflammatory cytokines,

like TNF- and IL-1, their secretion of chemokines and their ability to phagocytose invading

pathogens. Macrophages, which have exceptionally high phagocytic properties, destroy

ingested pathogens by digesting them. For that purpose, phagosomes fuse with lysosomes,

which have a low pH and are filled with enzymes, nitric oxide (NO) and reactive oxygen

species (ROS) (Michael Ehrenstein et al., 2008b; Michael Ehrenstein et al., 2008a; Dale et al.,

2008). Dendritic cells promote adaptive immune responses by presenting antigens derived

from ingested pathogens to T cells and thereby activating them (Leon et al., 2007).

Introduction

13

FIG. 4. Mononuclear phagocyte. Mononuclear phagocytes express the Fc receptor CD64, as well as CD14 and

TLR-4 which bind and recognize bacterial lipopolysaccharide (LPS). They secrete pro-inflammatory cytokines

like TNF- and IL-1 and contain lysosomes which serve for digestion of pathogens.

In mouse models researchers have investigated the role of macrophages in viral infections and

subsequently demonstrated the importance of macrophages in innate resistance to viruses

(Mogensen, 1979). In the case of HSV infection macrophages are among the first immune

cells to be activated and to exert antiviral activity (Ellermann-Eriksen, 2005). They can be

infected by HSV, but are non- or barely permissive for viral replication, depending on their

state of differentiation (Bruun et al., 1998; Daniels et al., 1978). Macrophages of HSV-

infected mice have been demonstrated to exert extrinsic antiviral activity in vitro, thereby

limiting viral replication in cell culture, independently of the virus or the host cell species

used (Morahan et al., 1980). The observed extrinsic antiviral activity is due to various

cytokines and anti-microbial molecules secreted by HSV-activated macrophages, like

IFN-/, ROS and NO, which directly inhibit HSV replication. Other cytokines like TNF-

and IL-12 activate other immune cells like NK cells (Voth et al., 1988; Wolf et al., 1991). In

HSV-infected mice mononuclear phagocytes are among the first cell populations recruited to

the infection site (Frank et al., 2012). They limit viral replication via TNF- secretion and NO

production (Fields et al., 2006; Kodukula et al., 1999) and are also required for the

development of an adaptive immune response (Cheng et al., 2000). Monocytes and

macrophages furthermore serve as important accessory cells in NK cell activation not only by

HSV-1 but by diverse viral, bacterial, and also protozoan pathogens. They activate NK cells

Introduction

14

via secretion of various cytokines, like IL-12, IL-15 and IL-18, as well as direct cell contact

through different receptor-ligand interactions, like natural killer group 2, member D

(NKG2D)-MHC class I polypeptide-related sequence (MIC) A or NKG2D-UL-16-binding

proteins (ULBP), natural cytotoxicity triggering receptor 1 (NKp46)-DNAX accessory

molecule-1 (DNAM1), and 2B4-CD48, depending on the respective pathogen (Michel et al.,

2012).

Introduction

15

2.5 Interactions of PDC and NK cells in HSV infection

Interactions between PDC and NK cells in HSV infection have been investigated in vivo and

in vitro. Barr et al. described PDC-NK cell interactions via IL-18 as important for NK cell

IFN- secretion after HSV-1 infection in mice. However, PDC were not the only cell

population activating NK cells, and CD69 expression as well as cytotoxicity of NK cells was

independent of IL-18 (Barr et al., 2007). Feldman et al. showed a role for accessory cells

(AC) in human NK cell-mediated lysis of HSV-1-infected fibroblasts: NK cell cytotoxicity

against infected fibroblasts was only accomplished in the presence of AC, which were, at least

in part, so called interferon producing cells (IPC), later identified as PDC. Participation of AC

was described to be IFN--dependent as well as IFN--independent, and probably cell

contact-dependent (Feldman et al., 1992). Another study demonstrated the in vitro ability of

HSV-1-stimulated PDC to induce migration of NK cells via secretion of chemokine (C-C

motif) ligand (CCL)4 and chemokine (C-X-C motif) ligand (CXCL)10 (Megjugorac et al.,

2004). A mouse study conducted by Persson et al. showed that HSV-1-stimulated PDC

recruited and activated NK cells in vivo (Persson and Chambers, 2010), and Swiecki et al.

demonstrated a critical role for PDC in NK cell activation in systemic HSV-1 and HSV-2

infections in mice (Swiecki et al., 2013). In a study of recurrent human HSV-2 infection, PDC

and NK cells co-localized in recurrent genital herpes lesions (Donaghy et al., 2009).

Several studies of human PDC-NK cell interaction after stimulation with CpG

oligodeoxynucleotides (CpG-ODN) indicated a major role for cytokines, particularly IFN-,

and a minor role for direct cell contact. In all studies CD69 expression on NK cells was cell

contact-independent but dependent on IFN- and other soluble factors like TNF-(Gerosa et

al., 2005; Benlahrech et al., 2009; Romagnani et al., 2005; Marshall et al., 2006). Cytotoxicity

was described to be induced by either soluble factors alone (Gerosa et al., 2005; Romagnani et

al., 2005) or demanded direct cell contact (Benlahrech et al., 2009), while IFN- secretion

induced by PDC was reported to be cytokine-mediated (Benlahrech et al., 2009; Romagnani

et al., 2005; Marshall et al., 2006).

Rationale

16

3 Rationale

Altogether, studies of NK cell activation in human HSV-1 infections, and particularly NK cell

interactions with potential accessory cells, like PDC, are limited, and the so far existing data

are controversial and insufficient. There are only few in vitro studies concerning human NK

cell activation by HSV-1, and in vivo studies which were mostly conducted in mice. In most

in vitro studies investigating NK cell-PDC interaction CpG-ODN were used as surrogate for

DNA viruses like HSV-1, but these studies only cover stimulatory effects of viral DNA, not

of other components of the HSV-1 particle nor the possible impact of viral replication.

Therefore, the goal of this study was to analyze the potential of HSV-1 to activate human NK

cells in vitro within the PBMC context, and to decipher mechanisms leading to HSV-1-

induced NK cell activation, in particular PDC-NK cell interactions, to identify further

accessory cell populations interacting with NK cells, and to determine cytokines involved in

the cellular crosstalk between NK cells and accessory cells. For these purposes, sucrose

gradient-purified UV-inactivated (HSVUV) as well as infectious (HSVINF) HSV-1 were used to

stimulate primary human PBMC.

Materials and Methods

17

4 Materials and Methods

4.1 Materials

4.1.1 Instruments

Instrument Manufacturer

BD LSRII BD Biosciences (Heidelberg, DE)

Biogard hood The Baker Company (Sanford, ME, US)

Bio-Link 254 UV crosslinker Vilber Lourmat (Eberhardzell, DE)

Eclipse TS 100 inverted microscope Nikon (Düsseldorf, DE)

ELx800 Absorbance Microplate Reader BioTek (Bad Friedrichshall, DE)

Finnpipette 300µl multi channel pipet Thermo scientific (Langenselbold, DE)

Heraeus Labofuge M Thermo scientific (Langenselbold, DE)

L7-55 ultracentrifuge Beckman Coulter (Krefeld, DE)

Micro 200R centrifuge Hettich lab technology (Tuttlingen, DE)

Neubauer Chamber hemocytometer Marienfeld Superior (Lauda-Königshofen, DE)

Pipetman 20µl pipet Gilson (Middleton, WI, US)



pipetus electrical pipette filler Hirschmann (Eberstadt, DE)

Reax top vortexer Heidolph (Schwabach, DE)

Research plus 20µl pipet Eppendorf (Wesseling-Berzdorf, DE)



Research 100µl multi channel pipet Eppendorf (Wesseling-Berzdorf, DE)

Rotina 380R centrifuge Hettich lab technology (Tuttlingen, DE)

Rotilabo mini centrifuge Roth (Karlsruhe, DE)

Stericult 200 incubator Labotect (Göttingen, DE)

SW 32Ti rotor Beckman Coulter (Krefeld, DE)

Thermomixer comfort 2ml Eppendorf (Wesseling-Berzdorf, DE)


18

4.1.2 Consumables

Consumable Manufacturer

0.5ml Micro-tubes Roth (Karlsruhe, DE)

1.5ml Micro-tubes Brand (Wertheim, DE)

1.5ml Screw cap micro tubes Sarstedt (Nümbrecht, DE)

2ml Micro-tubes Sarstedt (Nümbrecht, DE)

5ml FACS tubes Sarstedt (Nümbrecht, DE)

15ml centrifuge tubes Sarstedt (Nümbrecht, DE)

38.5ml polyallomer tubes Beckman Coulter (Krefeld, DE)

38.5ml ultra clear tubes Beckman Coulter (Krefeld, DE)

50ml centrifuge tubes Sarstedt (Nümbrecht, DE)

caps for FACS tubes Sarstedt (Nümbrecht, DE)

Cellstar filtertop cell culture flasks 650ml Greiner bio-one (Solingen, DE)



Cellstar 24 well plates Greiner bio-one (Solingen, DE)

Cellstar 96 well plates Greiner bio-one (Solingen, DE)

Cellstar cell culture plates 100mm Greiner bio-one (Solingen, DE)

Cellstar cell culture plates 60mm Greiner bio-one (Solingen, DE)

Costar 5mL Stripette, Polystyrene Corning (Wiesbaden, DE)



filter, 0.22µm BD Biosciences (Heidelberg, DE)

microscope cover slips Menzel-Gläser (Braunschweig, DE)

Nunc MaxiSorp 96 well plates Thermo scientific (Langenselbold, DE)

paper towels Tork (Mannheim, DE)

Pipet tips 1000µl Ratiolab (Dreieich, DE)

Pipet tips 200µl Sarstedt (Nümbrecht, DE)

SafeGuard filter tips 1250µl Peqlab (Erlangen, DE)

Safety Multifly needle Sarstedt (Nümbrecht, DE)

Silver Nitrile gloves S Kimberly-Clark (Koblenz, DE)

S-Monovette EDTA K2 gel Sarstedt (Nümbrecht, DE)

Stericup Filter Unit, 0.22µm, 150ml Merck Millipore (Darmstadt, DE)



syringe, 10ml BD Biosciences (Heidelberg, DE)


19

4.1.3 Reagents

Reagent Manufacturer

acetic acid (C2H4O2) Merck Millipore (Darmstadt, DE)

Biocoll 1.077g/ml Biochrom (Tutzing, DE)

bovine serum albumin (BSA) Sigma-Aldrich (München, DE)

CpG-A 6016 (5´-T*C-G-A-C-G-T-C-G-T-G-

G*G*G*G-3´)

* stands for phosphorothioate

- stands for phosphodiester bonds

Coley Pharmaceutical (Düsseldorf, DE)

disodium phosphate (Na2HPO4) Merck Millipore (Darmstadt, DE)

Dulbecco`s Modified Eagle Medium (DMEM) Invitrogen (Darmstadt, DE)

ethylenediaminetetraacetic acid (EDTA) Sigma-Aldrich (München, DE)

fetal calf serum (FCS) Sigma-Aldrich (München, DE)

glucose Merck Millipore (Darmstadt, DE)

glutamine Invitrogen (Darmstadt, DE)

recombinant human interferon-2b (rhIFN-) Miltenyi Biotec (Bergisch Gladbach, DE)

recombinant human interleukin 2 (rhIL-2) Roche-Pharma (Grenzach-Wyhlen, DE)

recombinant human interleukin 3 (rhIL-3) R&D Systems (Wiesbaden-Nordenstadt, DE)

hydrogen chloride (HCl) Merck Millipore (Darmstadt, DE)

monopotassium phosphate (KH2PO4) Merck Millipore (Darmstadt, DE)

paraformaldehyde (PFA) Sigma-Aldrich (München, DE)

penicillin Invitrogen (Darmstadt, DE)

phenol red Merck Millipore (Darmstadt, DE)

potassium chloride (KCl) Merck Millipore (Darmstadt, DE)

Roswell Park Memorial Institute (RPMI) 1640

Medium

Invitrogen (Darmstadt, DE)

sodium chloride (NaCl) Merck Millipore (Darmstadt, DE)

streptomycin Invitrogen (Darmstadt, DE)

sulfuric acid (H2SO4) Merck Millipore (Darmstadt, DE)

tris(hydroxymethyl)aminomethane (Tris) Roth (Karlsruhe, DE)

trypan blue Sigma-Aldrich (München, DE)

Tween 20 Roth (Karlsruhe, DE)


20

4.1.4 Software

Software Source

FACSDiva Software BD Biosciences (Heidelberg, DE)

FCS Express 3 Software De Novo Software (Los Angeles, CA, US)

FlowCytomixPro software Affymetrix eBioscience (Frankfurt, DE)

Gen5 Data Analysis Software BioTek (Bad Friedrichshall, DE)

VassarStats Statistical Computation Website http://www.vassarstats.net/

4.1.5 Commercial Kits

Kit Manufacturer

Human CD304 MicroBead Kit Miltenyi Biotec (Bergisch Gladbach, DE)

Human CD14 MicroBeads Miltenyi Biotec (Bergisch Gladbach, DE)

Human IFN- Matched Antibody Pairs Affymetrix eBioscience (Frankfurt, DE)

Human sCD40L Matched Antibody Pairs Affymetrix eBioscience (Frankfurt, DE)

Human IFN- Secretion Assay Detection Kit Miltenyi Biotec (Bergisch Gladbach, DE)

Human NK Cell Isolation Kit Miltenyi Biotec (Bergisch Gladbach, DE)

Human Th1/Th2 11plex RTU FlowCytomix

Multiplex

Affymetrix eBioscience (Frankfurt, DE)

Human TNF- Secretion Assay Detection Kit Miltenyi Biotec (Bergisch Gladbach, DE)

4.1.6 Cell Culture

Cultured cells Cell type Origin

Human foreskin fibroblasts

(HFF)

neonatal foreskin Human

Primary blood cells peripheral blood mononuclear cells

(PBMC)

Human

Primary blood cells plasmacytoid dendritic cells (PDC) Human

Primary blood cells natural killer (NK) cells Human

Primary blood cells monocytes Human

Vero cells deficient for IFN-

and IFN-1 genes (Diaz et al.,

1988)

kidney epithelial cells African green monkey


21

4.1.7 Viruses

Virus Clone Source

Herpes simplex virus type 1

(HSV-1), expressing a green

fluorescent protein (GFP)-tagged

VP22

166v Gillian Elliott, Peter O’Hare (Elliott and

O'Hare, 1999)


(HSV-1), ICP4, ICP22, ICP27,

ICP47 deletion mutant,

expressing GFP under a HCMV

promoter

d106S David M. Knipe (Liu et al., 2009)


(HSV-1), wild type

primary isolate diagnostic services, Institute of Clinical

and Molecular Virology, Friedrich-

Alexander-University Erlangen-Nürnberg

(Kittan et al., 2007)

4.1.8 Media and Buffers

Medium / Buffer Composition

assay buffer (Matched Antibody Pairs) 0.5% BSA

0.05% Tween 20

in DPBS (Matched Antibody Pairs)

cytokine buffer (cytokine secretion assay) 0.5% BSA

2mM EDTA

in DPBS

coating solution (IFN- Matched Antibody Pairs) 1µg/ml antibody


coating solution (sCD40L Matched Antibody Pairs) 5µg/ml antibody


DPBS 138mM NaCl

2.7mM KCl

6.5mM Na2HPO4

1.5mM KH2PO4

DPBS (Matched Antibody Pairs) 8g NaCl

0.2g KCl

2.85g Na2HPO4 x12 H2O

0.2g KH2PO4

ad 1l H2O

FACS buffer 1% FCS

1mM EDTA

in DPBS

MACS buffer 1% FCS

2mM EDTA

in DPBS


22

Medium / Buffer Composition

stop solution (Matched Antibody Pairs) 4N H2SO4

supplemented RPMI 1640 0.3mg/ml glutamine

200U/ml penicillin

90U/ml streptomycin

10% FCS

supplemented DMEM 0.3mg/ml glutamine

200U/ml penicillin

90U/ml streptomycin

10% FCS

Trypsin EDTA 140mM NaCl

5mM KCl

0.65mM Na2HPO4

5mM glucose

25mM Tris/HCl

0.01% EDTA

0.1% phenole red

virus standard buffer (VSB) 0.05M Tris

0.012M KCl

0.005M EDTA

pH 7.8

VSB 15% sucrose solution 15% sucrose

0.1% BSA

in VSB

VSB 30% sucrose solution 30% sucrose

0.1% BSA

in VSB

washing buffer (Matched Antibody Pairs) 0.05% Tween 20

in DPBS

4.1.9 Antibodies

Epitope Flurophore Clone Isotype Manufacturer

CD1c FITC L161 mouse IgG1 Biolegend (London, GB)

CD3 FITC UCHT1 mouse IgG1 Biolegend (London, GB)

CD3 PE UCHT1 mouse IgG1 Biolegend (London, GB)

CD3 PE-Cy5 UCHT1 mouse IgG1 AbD Serotec (Düsseldorf, DE)

CD3 AlexaFluor700 UCHT1 mouse IgG1 Biolegend (London, GB)

CD3 PacificBlue UCHT1 mouse IgG1 BD Biosciences (Heidelberg, DE)

CD4 PE-Cy7 RPA-T4 mouse IgG1 Biolegend (London, GB)

CD8 APC MEM-31 mouse IgG2a ImmunoTools (Friesoythe, DE)


23

Epitope Flurophore Clone Isotype Manufacturer

CD8 APC-eFluor780 RPA-T8 mouse IgG1 Affymetrix eBioscience

(Frankfurt, DE)

CD14 PE-Cy5 61D3 mouse IgG1 AbD Serotec (Düsseldorf, DE)

CD16 PE-Cy7 3G8 mouse IgG1 Biolegend (London, GB)

CD19 APC HIB19 mouse IgG1 Biolegend (London, GB)

CD33 PE WM53 mouse IgG1 Biolegend (London, GB)

CD56 PE HCD56 mouse IgG1 Biolegend (London, GB)

CD56 PE-Cy7 HCD56 mouse IgG1 Biolegend (London, GB)

CD64 APC 10.1 mouse IgG1 Biolegend (London, GB)

CD69 FITC FN50 mouse IgG1 Miltenyi Biotec (Bergisch

Gladbach, DE)

CD69 AlexaFluor700 FN50 mouse IgG1 Biolegend (London, GB)

CD107a AlexaFluor488 eBioH4A3 mouse IgG1 Affymetrix eBioscience

(Frankfurt, DE)

CD123 PE AC145 mouse IgG2a Miltenyi Biotec (Bergisch

Gladbach, DE)

CD303 FITC AC144 mouse IgG1 Miltenyi Biotec (Bergisch

Gladbach, DE)

CD304 APC AD5-17F6 mouse IgG1 Miltenyi Biotec (Bergisch

Gladbach, DE)

HLA-ABC PE W6/32 mouse IgG2a Biolegend (London, GB)

HLA-E PE 3D12 mouse IgG1 Biolegend (London, GB)

IFN-/R none MMHAR-2 mouse IgG2a Acris (Herford, DE)

IFN- APC 45-15 mouse IgG1 Miltenyi Biotec (Bergisch

Gladbach, DE)

IL-1 none 8516 mouse IgG1 R&D Systems (Wiesbaden-

Nordenstadt, DE)

MICA /

MICB

APC 6D4 mouse IgG2a Biolegend (London, GB)

TNF- PE cA2 human IgG1 Miltenyi Biotec (Bergisch

Gladbach, DE)

TNF- none 28401 mouse IgG1 R&D Systems (Wiesbaden-

Nordenstadt, DE)


24

4.1.10 Isotype Controls

Flurophore Clone Isotype Manufacturer

AlexaFluor700 MOPC-21 mouse IgG1 Biolegend (London, GB)

APC MOPC-21 mouse IgG1 Biolegend (London, GB)

APC PPV-04 mouse IgG2a ImmunoTools (Friesoythe, DE)

APC-eFluor780 MOPC-21 mouse IgG1 Biolegend (London, GB)

FITC MOPC-21 mouse IgG1 Biolegend (London, GB)

none 11711 mouse IgG1 R&D Systems (Wiesbaden-

Nordenstadt, DE)

none PPV-04 mouse IgG2a Acris (Herford, DE)

PacificBlue MOPC-21 mouse IgG1 BD Biosciences (Heidelberg, DE)

PE MOPC-21 mouse IgG1 Biolegend (London, GB)

PE MOPC-173 mouse IgG2a Biolegend (London, GB)

PE-Cy5 MCA928C mouse IgG1 AbD Serotec (Düsseldorf, DE)

PE-Cy7 MOPC-21 mouse IgG1 Biolegend (London, GB)


25

4.2 Methods

4.2.1 Isolation of primary human cells

Peripheral blood mononuclear cells

PBMC were isolated from EDTA-anti-coagulated blood of healthy donors using Biocoll

density centrifugation (1.077g/ml). These studies were approved by the Ethical Committee of

the Medical Faculty, Friedrich-Alexander-Universität Erlangen-Nürnberg (Ref. no. 3299).

EDTA blood was centrifuged at 200x g for 10min and plasma was removed. Cells of four

vials were then transferred into a 50ml tube, filled up to 35ml with RPMI 1640 and layered

onto 15ml Biocoll. Separation of PBMC from erythrocytes and granulocytes was achieved by

centrifugation at 440x g for 25min with the brake inactivated. The interphase containing

lymphocytes and monocytes, visible as a ring between the upper cell-free layer and the

Biocoll layer, was transferred into a 50ml tube, filled up to 50ml with RPMI 1640 and

centrifuged at 440x g for 5min. This washing step was repeated, and cells were then re-

suspended in supplemented RPMI 1640. Cell numbers were determined using a Neubauer

chamber.

Plasmacytoid dendritic cells

PDC were isolated from PBMC via magnetic-activated cell sorting (MACS) using the CD304

MicroBead Kit (Miltenyi Biotec). PBMC were centrifuged at 440x g for 5min. Supernatant

was discarded and cells were washed by re-suspension in MACS buffer and centrifugation at

440x g for 5min. Cells were then re-suspended in MACS buffer and incubated with FcR

blocking reagent and CD304 MicroBeads at 4°C for 15min. Per one million cells, 1.5µl

MACS buffer, 0.5µl FcR blocking reagent and 0.5µl CD304 MicroBeads were used. Cells

were then washed, re-suspended in 1ml MACS buffer and applied to a LS MACS column that

had been placed in a MACS separator and equilibrated with 3ml MACS buffer. Magnetically

labeled PDC were retained within the column, while unlabeled cells could flow through. After

three washing steps with 3ml MACS buffer, the column was removed from the separator and

PDC were eluted using 10ml MACS buffer. Flow through was used as PDC-depleted PBMC

for depletion experiments. After centrifugation a second round of isolation followed using a

MS MACS column and volumes of 500µl for equilibration, re-suspension and washing and

4ml for elution. Numbers of purified PDC were determined using a Neubauer chamber.

Monocytes

Monocytes were isolated from PBMC via MACS using CD14 MicroBeads (Miltenyi Biotec).

PBMC were centrifuged at 440x g for 5min, supernatant was discarded and cells were washed

by re-suspension in MACS buffer and centrifugation at 440x g for 5min. Cells were then re-


26

suspended in MACS buffer and incubated with CD14 MicroBeads at 4 °C for 15min. Per one

million cells, 4µl MACS buffer and 1µl CD14 MicroBeads were used. Cells were then

washed, re-suspended in 1ml MACS buffer and applied to a LS MACS column that had been

placed in a MACS separator and equilibrated with 3ml MACS buffer. Magnetically labeled

monocytes were retained within the column, while unlabeled cells could flow through. After

three washing steps with 3ml MACS buffer, the column was removed from the separator and

monocytes were eluted using 10ml MACS buffer. Flow through was used as

monocyte-depleted PBMC for depletion experiments. Numbers of purified monocytes were

determined using a Neubauer chamber.

Natural killer cells

NK cells were isolated from PBMC via MACS using the NK Cell Isolation Kit (Miltenyi

Biotec). PBMC were centrifuged at 440x g for 5min, supernatant was discarded and cells

were washed by re-suspension in MACS buffer and centrifugation at 440x g for 5min. Cells

were then re-suspended in MACS buffer and incubated with a biotinylated antibody cocktail

against lineage markers of non-NK cells at 4°C for 10min. Per one million cells, 4µl MACS

buffer and 1µl antibody cocktail were used. Cells were then further incubated with

MicroBead-coupled secondary antibodies directed against biotin at 4°C for 15min, using 3µl

MACS buffer and 2µl secondary antibodies per one million cells. Cells were then washed,

re-suspended in 1ml MACS buffer and applied to a LS MACS column that had been placed in

a MACS separator and equilibrated with 3ml MACS buffer. Magnetically labeled non-NK

cells were retained within the column, while unlabeled NK cells could flow through.

Thereafter, three washing steps with 3ml MACS buffer were performed. Numbers of purified

NK cells were determined using a Neubauer chamber.

4.2.2 Determination of cell numbers

For determination of PBMC numbers, one volume of cell suspension was mixed with one

volume of Turks solution (3% C2H4O2) to lyse erythrocytes still present within PBMC, and

with two volumes of Trypan blue to exclude dead cells from the count. This resulted in a 1:4

dilution of the cells. A Neubauer chamber was filled with the suspension, and cells were

counted within four sixteen-square fields, of which one is equivalent to 0.1µl.

The number of cells per ml was calculated as depicted below.

cells / ml = cells counted x104

For counting of purified cells or cell lines, one volume of cell suspension was mixed with one

volume Trypan blue, leading to a 1:2 dilution of cells, and cells were counted within two

sixteen-square fields.


27

FIG. 5. Sixteen-square field of a Neubauer chamber. The cell number counted within this sixteen-square field

is equivalent to the cell number per 0.1µl and is multiplied with 104 to obtain the cell number per ml.

4.2.3 Herpes simplex virus type 1 stocks

Generation

For the generation of HSV-1 stocks, Vero cells were cultured in 650ml cell culture flasks. A

confluent monolayer was inoculated with HSV-1 stock in a volume of 20ml per cell culture

flask. After incubation at 37°C for 2h, cell cultures were washed with 50ml warm DMEM and

then given 50ml supplemented DMEM per tissue culture flask. After incubation at 37°C for

3d, infected cells were re-suspended. After two freeze-and-thaw cycles, lysates were

centrifuged at 440x g for 5min and supernatants were harvested. Cell-free supernatants were

either purified over a sucrose gradient or used directly. Directly used lysates were filtered

through a 0.22µm filter. Aliquots were stored at -80°C.

Purification

Cell-free supernatants were filled into 38.5ml polyallomer tubes and centrifuged in an

ultracentrifuge at 50,000x g for 90min at 4°C. Supernatants were discarded; virus pellets were

incubated in the residual liquid overnight at 4°C, re-suspended and pooled and dounced

twenty times. A continuous gradient from 30% to 15% sucrose was filled into a 38.5ml ultra

clear tube, re-suspended virus was layered upon the sucrose gradient and centrifuged at

50,000x g for 30min at 4°C. The virus ring that was visible within the sucrose gradient when

exposed to strong light of a microscope was collected, given into a 38.5ml polyallomer tube,

filled up to 38.5ml with virus standard buffer and centrifuged at 78,000x g for 90min at 4°C.

Supernatant was discarded; the virus pellet was incubated in the residual liquid for 1h at 4°C,

re-suspended in RPMI 1640 and filtered through a 0.22µm filter (HSVINF). Part of he purified

virus stock was inactivated by exposure to ultraviolet light (HSVUV). Aliquots were stored at

-80°C.

UV-inactivation

Virus stocks were inactivated using an UV crosslinker (Vilber Lourmat). They were irradiated

in an open cell culture plate five times to a final dose of 1J/cm2, with shaking of the plate

between irradiation rounds. Complete UV-inactivation was proven by inoculation of Vero

cells with undiluted virus stock resulting in lack of cytopathic effect and hence lack of viral

infection in the cell culture after 3d of incubation at 37°C.


28

Determination of the TCID50/ml

For the determination of the 50% tissue culture infective dose (TCID50)/ml of a virus stock,

Vero cells from one 50ml cell culture flask were re-suspended in 25ml supplemented DMEM

and seeded into three 96 well plates using 75µl per well. The virus stock was pre-diluted

1:100, 1:1,000 and 1:10,000 for the first, second and third plate, respectively, and a 1:4

dilution series was achieved for each pre-dilution by pipetting 25µl pre-diluted virus stock

into the eight wells of the first row, mixing, pipetting 25µl from the first row into the second

row and so on, until the last row of each plate was filled. Cell cultures were incubated at 37°C

for 3d and then screened for cytopathic effect indicating infection. The TCID50/ml was

calculated according to the method of estimating fifty percent endpoints published by Reed

and Muench (L.J.REED and H.MUENCH, 1938). For this purpose, the threshold between the

last row with 50% or more infected wells and the first row with less than 50% infected wells

was determined for each 96 well plate. The TCID50/ml for each plate was calculated as

depicted below and a mean TCID50/ml was determined.

e = ( a / ( a + b ) ) x100

f = ( c / ( c + d ) ) x100

P = ( e - 50% ) / ( e - f )

TCID50 / ml = D R + P

x C x ( 1 / V)

a: (total of infected wells below the threshold and in the last row above the threshold) x2

b: (total of non-infected wells above the threshold) x2

c: (total of infected wells below the threshold) x2

d: (total of non-infected wells above the threshold and in the first row below the threshold) x2

e: percentage of infected wells above the threshold

f: percentage of infected wells below the threshold

D: applied dilution series of virus stock

R: last row above the threshold

P: proportional distance

V: applied volume of virus stock (ml)

C: applied pre-dilution of virus stock

4.2.4 PDC supernatants

Generation

PDC supernatants (PDC-SN) were generated by stimulation of PDC with HSVINF. A total of

5x105 PDC were cultured in 500µl supplemented RPMI 1640 containing 20ng/ml rhIL-3 in

24 well plates and inoculated with 1x106 TCID50/ml HSV-1. After incubation at 37°C for 3h

PDC were harvested and centrifuged at 590x g for 10min. PDC were washed with DPBS, re-


29

suspended in 100µl trypsin EDTA, and after incubation at 37°C for 15min, PDC were washed

again and cultured in 500µl supplemented RPMI 1640 containing 20ng/ml rhIL-3 at 37°C for

18h. PDC-SN were then harvested and stored at -20°C. IFN-2a/2b concentrations were

determined using the IFN- Matched Antibody Pairs (Affymetrix eBioscience).

Determination of inhibitory potential on HSV-1 replication

In order to determine the potential of PDC-SN to inhibit HSV-1 replication in target cells,

human foreskin fibroblasts (HFF) were cultured in 24 well plates, using 1x105 cells in 500µl

supplemented DMEM per culture, and inoculated with a green fluorescing HSV-1 (HSVGFP)

at an MOI of 0.01 and 0.001. After incubation at 37°C for 2h, virus-containing media of cell

cultures were exchanged with fresh media containing either no PDC-SN or PDC-SN at

concentrations of different IFN-2a/2b concentrations. After incubation at 37°C for 24h and

48h, cells were harvested for FACS analysis. Cells were analyzed for infection and viability.

Stimulation of NK cells

A total of 2.5x105 NK cells were cultured in 24 well plates in 500µl supplemented RPMI

1640, inoculated with PDC-SN or recombinant human IFN-2b (rhIFN-) (Invitrogen), using

comparable IFN-2a/2b concentrations, incubated at 37°C for 3 to 18h and harvested for

FACS analysis. For neutralization of type I IFN activity, 15µg/ml anti ()IFN-/ receptor

(IFN-R) antibody was added to the cell culture. Activation of cells was determined by

surface expression of CD69.

4.2.5 Stimulation and infection of cells with HSV-1

Stimulation of PBMC

A total of 1x106 PBMC or PBMC depleted of monocytes or PBMC depleted of PDC were

cultured in 24 well plates in 500µl supplemented RPMI 1640 and inoculated with 1x106

TCID50/ml HSVUV and HSVINF, 0.75µM CpG-A, and 100U/ml rhIL-2. Mock served as

control. For neutralization experiments, IL-1, TNF-, IFN-R, and their respective

isotype controls were added to cell cultures at a concentration of 15µg/ml before stimulation.

PBMC were incubated for 12 to 18h at 37°C and then harvested for FACS analysis. Activated

cells were determined by surface expression of CD69, by degranulation and by secretion of

IFN- and TNF-.

Stimulation of NK cells in the presence of PDC and HFF

A total of 2x105 NK cells were cultured in 24 well plates in 500µl supplemented RPMI 1640

without any further cell population, with PDC in the donor-specific ratio to NK cells, with

1x105 HFF or with both PDC and HFF. Cell cultures were inoculated with 1x10

6 TCID50/ml


30

HSVUV and HSVINF, mock served as control. After incubation at 37°C for 24h, cells were

harvested for FACS analysis. NK cells were analyzed for surface expression of CD69 and

CD56.

Infection of monocytes

A total of 5x105 monocytes were cultured in 24 well plates in 500µl supplemented RPMI

1640, inoculated with HSVGFP, HSVUV and HSVINF at a MOI of 1, incubated at 37°C for 24

and 48h and harvested for FACS analysis. For neutralization experiments, IFN-R and an

isotype control were added to cell cultures at a concentration of 15µg/ml before inoculation.

Cells were analyzed for infection, viability and expression of lineage markers and monocyte

markers, as well as MHC class I (MHC-I) molecules and stress-induced molecules.

Infection of cells for quantitative polymerase chain reaction (PCR)

A total of 1x105 monocytes and HFF were cultured in 24 well plates in 500µl supplemented

RPMI 1640 and DMEM, respectively, and inoculated with HSVGFP or an infectious, but non-

replicative HSV-1 variant (HSVd106S) at a MOI of 1. After incubation at 37°C for 2h, cells

were washed once with DPBS, incubated with 100µl trypsin EDTA at 37°C for 10min and

then re-suspended to separate cells from each other and from the well surface. After addition

of 100µl supplemented cell culture medium cells were centrifuged at 590x g for 10min, re-

suspended in 500µl supplemented cell culture medium and cultured in fresh 24 well plates.

After incubation at 37°C for 24, 48, 72 and 120h, supernatants were harvested and stored at

-20°C.

4.2.6 FACS analysis of cells

Degranulation assay

For the determination of degranulation, 5µl fluorescing CD107a antibody per cell culture

was added 1.5h before harvesting the cells. In case of degranulation, the antibody could bind

to CD107a, which is a protein lining the membranes of endosomal and secretory vesicles and

being temporarily exposed on the surface of a degranulating cell.

Harvesting of cells

Primary cells were put on ice for 10min and then re-suspended thoroughly to remove all cells

from the well surface. HFF were washed once with DPBS, incubated with 100µl trypsin

EDTA at 37°C for 10min and then re-suspended to separate cells from each other and from

the well surface.


31

Cytokine secretion assays

Secretion of IFN- and TNF- by PBMC was determined using the IFN- Secretion Assay

Detection Kit and the TNF- Secretion Assay Detection Kit (Miltenyi Biotec). Harvested

cells were centrifuged at 590x g for 10min, washed once with 900µl cold cytokine buffer, re-

suspended in 90µl cold supplemented RPMI 1640 and after addition of 10µl cytokine catch

reagent antibody incubated on ice for 5min. After addition of 900µl warm supplemented

RPMI 1640, cells were incubated at 37°C for 45min in a micro tube shaker. Thereafter, cells

were put on ice for a few seconds, 1ml of cold cytokine buffer was added, and cells were

centrifuged and labeled for FACS analysis, starting with the blocking step.

Labeling of cells for FACS analysis

Harvested cells were centrifuged at 590x g for 10min, washed once with 900µl FACS buffer,

then re-suspended in 100µl FACS buffer and incubated at 4°C for 10min in the presence of

3µl FcR blocking reagent. Blocked cells were incubated with antibodies against specific cell

surface and activation markers at 4°C for 20min, washed with 3ml FACS buffer and re-

suspended in 100 - 180µl 4% PFA for fixation.

Live-dead staining of cells

Viability of cells was determined using a Fixable Violet Dead Cell Stain Kit (Invitrogen).

Cells were washed once with FACS buffer, resuspended in 100µl FACS buffer and incubated

with 0.5µg dye at 4°C for 20min, washed with 3ml FACS buffer and re-suspended in 100 -

180µl 4% PFA for fixation.

FACS analysis

Cells were analyzed in an LSRII (BD Biosciences), equipped with FACSDiva Software (BD

Biosciences) for automatic compensation and measurement of probes. Results of the

measurements were evaluated using the FCS Express 3 Software (De Novo Software).

4.2.7 Determination of secreted cytokines within supernatants

IFN-2a/2b

IFN-2a/2b (IFN-) concentrations were determined using the Human IFN- Matched

Antibody Pairs (Affymetrix eBioscience). Microwell plates were coated with 100µl coating

solution per well, sealed with an adhesive cover and incubated at 4°C overnight. After

washing once with 300µl washing buffer per well, the plate was blocked with 200µl assay

buffer per well, sealed with an adhesive cover and incubated either at room temperature for 2h

or at 4°C overnight. Standard was prepared by dilution of concentrated standard protein with

assay buffer to reach 1ng/ml standard protein. HRP-conjugate was prepared by dilution of


32

5.5µl concentrated HRP-conjugate with assay buffer to a final volume of 5.5ml. The

microwell plate was washed twice with 300µl washing buffer and filled with assay buffer:

Wells of rows 1 and 2 were filled with 100µl assay buffer for standard dilution and all other

wells were filled with 50µl assay buffer and 50µl probe. Samples were appropriately diluted

to measure within the linear range of the assay. A 1:2 dilution series of standard protein in

rows 1 and 2 was achieved by pipetting 100µl of 1ng/ml standard into the first two wells,

mixing the first dilution and pipetting 100µl of it into the next two wells. This procedure was

repeated until the penultimate wells were reached, 100µl of the last dilution were discarded,

and the last two wells filled with assay buffer served as blank controls. After addition of 50µl

HRP-conjugate per well, the plate was sealed with an adhesive cover and incubated at room

temperature for 2h on a microplate shaker. Substrate solution was prepared 30min before

continuation of the protocol by mixing equal volumes of H2O2 and tetramethylbenzidine.

After washing the plate three times, 100µl substrate solution per well was added and the plate

was incubated at room temperature for about 10min, avoiding direct exposure to light and

monitoring the color development of the standard. When the standard with the highest

concentration had developed a dark blue color, 100µl stop solution per well was added and

the plate was measured at 450nm with reference at 650nm.

sCD40L

sCD40L concentrations were determined using the Human sCD40L Matched Antibody Pairs

(Affymetrix eBioscience). Microwell plates serving as sample plates were coated with 100µl

coating solution per well, covered with an adhesive film and incubated at 4°C overnight. After

washing once with 300µl washing buffer per well, the sample plate was blocked with 200µl

assay buffer per well, sealed with an adhesive cover and incubated either at room temperature

for 2h or at 4°C overnight. Standard was prepared by dilution of concentrated standard protein

with assay buffer to reach 20ng/ml standard protein. HRP-conjugate was prepared by dilution

of 11µl concentrated HRP-conjugate with assay buffer to a final volume of 11ml. Wells of

rows 1 and 2 of a dilution plate were filled with 100µl sample diluent for further standard

dilution and all other wells were filled with 80µl sample diluent and 20µl of 1:5-diluted

plasma probes. A 1:2 dilution series of standard protein was achieved by pipetting 100µl of

20ng/ml standard into the first two wells, mixing the first dilution and pipetting 100µl of it

into the next two wells. This procedure was repeated until the penultimate wells were reached,

100µl of the last dilution were discarded, and the last two wells filled with sample diluent

served as blank controls. Last, 100µl HRP-conjugate were added per well. The sample plate

was washed twice with 300µl washing buffer. After transfer of 150µl from the wells of the

dilution plate into the sample plate, the sample plate was sealed with an adhesive cover and

incubated at room temperature for 2h on a microplate shaker. Substrate solution was prepared

30min before continuation of the protocol by mixing equal volumes of H2O2 and

tetramethylbenzidine. After washing the plate three times, 100µl substrate solution per well


33

was added and the plate was incubated at room temperature for about 10min, avoiding direct

exposure to light and monitoring the color development of the standard. When the highest

standard had developed a dark blue color, 100µl stop solution per well was added and plate

was measured at 450nm with reference at 650nm.

Th1/Th2 cytokines

Cytokines secreted into supernatants were analyzed using the Th1/Th2 11plex RTU

FlowCytomix Multiplex kit (Affymetrix eBioscience). For 96 samples, assay buffer was

prepared by dilution of 50ml 10x assay buffer with deionized H2O to a final volume of 500ml.

Bead mix was prepared by dilution of 1.5ml 2x bead mix with reagent dilution buffer to a

final volume of 3ml. Bead mix was centrifuged at 3,000x g for 5min, supernatant was

discarded, beads were re-suspended in 3ml reagent dilution buffer and mixed well. Biotin

conjugate mix was prepared by dilution of 3.5ml 2x biotin conjugate mix with reagent

dilution buffer to a final volume of 7ml. Standard protein was reconstituted in 200µl assay

buffer, mixed well and completely resolved within 10 - 30min. A 1:3 standard dilution series

was achieved by mixing 50µl standard with 100µl assay buffer, repeating this procedure with

the resulting dilution five times. Assay buffer served as negative control. FACS tubes were

filled with 25µl of the samples, standard dilution series and negative control. Standard

dilution series and negative control were used in duplicate, the standard with the highest

concentration was used in triplicate for instrument setup. After addition of 25µl bead mix and

50µl biotin conjugate mix per tube, probes were incubated at room temperature in the dark for

2h. Streptavidin-PE solution was prepared by dilution of 200µl concentrated streptavidin-PE

with 6,050µl assay buffer. After two washing steps of the probes (addition of 1ml assay buffer

per tube, centrifugation at 355x g for 5min and discarding of the supernatant), 50µl

streptavidin-PE solution per tube was added and probes were incubated at room temperature

in the dark for 1h. After two more washing steps, 500µl assay buffer was added per tube and

probes were ready for FACS analysis. Instrument was setup using the setup beads and the

standard with the highest concentration to adjust FSC / SSC parameters, to create regions for

the different bead populations, that were defined by different size and APC fluorescence

intensity, and to adjust voltage of PE emission, so that the bead population of the negative

control was visible far left in the plot, while the bead population of the highest standard was

visible far right in the plot. After analysis of the standard, probes were measured. Results of

the measurement were evaluated using the FlowCytomixPro software (Affymetrix

eBioscience).


34

4.2.8 Quantification of HSV-1 DNA

Isolation of viral DNA from cell culture supernatants

HSV-1 DNA was extracted from cell culture supernatants using the EZ1 Virus Mini Kit v2.0

together with the EZ1 Advanced XL robotic workstation (both Qiagen, Hilden, DE) according

to the manufacturer’s recommendations. A total of 200µl of supernatant was used for

extraction and DNA was eluted into 120µl of volume. Isolation of HSV-1 DNA was

performed by the diagnostic services of the Institute of Microbiology and Hygiene,

Regensburg.

Quantitative PCR

Absolute quantification of HSV-1 DNA was performed by realtime amplification of a

sequence within the HSV-1 glycoprotein G. HSV-1 DNA concentration within each sample

was determined with reference to standard controls containing defined copies of HSV-1 DNA.

The mastermix contained forward and reverse primers and VIC-/FAM-TAMRA-labeled

Taqman probes for HSV-1 and HSV-2 (Metabion, Martinsried, DE). 5µl of each sample was

added to 25µl mastermix and amplified in duplicates. Samples were analyzed using the

StepOnePlus Real-Time PCR System (Applied Biosystems, Darmstadt, DE). Initial

denaturation at 95°C for 10min was followed by 45 cycles of annealing and extension at 60°C

for 1min and denaturation at 95°C for 15sec. Quantification of HSV-1 DNA was performed

by the diagnostic services of the Institute of Microbiology and Hygiene, Regensburg.

4.2.9 Virological analysis of hyperproliferative lesions

A swab from the hyperproliferative lesions was analyzed using the RealArt HSV-1/2 PCR kit

according to the manufacturer’s recommendations (Qiagen). For the analysis of

papillomavirus DNA, E1 consensus primers were used for amplification, sequencing, and

GenBank alignement (Iftner et al., 2003). All analyses were performed by the diagnostic

services of the Institute of Clinical and Molecular Virology, Erlangen, Germany. These

studies together with the immunological analyses of the patient’s PBMC were approved by

the Ethical Committee of the Medical Faculty, Friedrich-Alexander-Universität Erlangen-

Nürnberg (Ref. no. 3375).

4.2.10 Statistical analysis

Statistical analysis was carried out using the online tool VassarStats Statistical Computation

(http://www.vassarstats.net/). For comparison of two samples the Student’s t-test was applied,

for comparison of three or more samples the Tukey HSD test was applied to account for

multiple comparisons.

Results

35

5 Results

5.1 Stimulation of PBMC with HSV-1 leads to NK cell activation

In order to analyze the potential of HSV-1 to induce NK cell activation and effector functions

within the PBMC context, we stimulated PBMC with infectious (HSVINF) and UV-inactivated

(HSVUV) HSV-1, CpG-A-ODN (CpG-A), a toll-like receptor 9 (TLR-9) agonist, and IL-2

(FIG. 6A). CpG-A served as representative of PDC-dependent NK cell activation (Hemmi et

al., 2000) and IL-2 was used for direct NK cell activation (Trinchieri et al., 1984). All stimuli

significantly up-regulated the activation marker CD69 on NK cells compared to the mock

control (p<0.01) (FIG. 6B, C). Comparison of two different time points displayed diverse

kinetics of NK cell activation by the different stimuli. At 12h post stimulation (p.s.) HSVINF

induced significantly stronger NK cell CD69 up-regulation than all other stimuli, while at 18h

p.s. the significant difference to HSVINF was lost for CpG-A and HSVUV, and reduced for

IL-2 (p<0.05). Altogether, the data indicate faster NK cell activation by HSVINF than by the

other stimuli.

All stimuli activated both CD56dim

and CD56bright

NK cells significantly compared to the

mock control at 12h and 18h p.s. (p<0.01), but activation of the CD56bright

population varied

between stimuli. CD56dim

cells were activated faster by HSVINF than by any other stimulus

(FIG. 7A), but significant differences between HSVINF and CpG-A as well as HSVUV did not

persist (FIG. 7C). IL-2 proved to be the strongest stimulus for CD69 up-regulation on

CD56bright

NK cells with significant differences to all other stimuli at both time points

(p<0.01) (FIG. 7B, D). Both HSVINF and HSVUV activated CD56bright

NK cells to a greater

extent than CpG-A, but the difference to CpG-A persisted only for HSVINF at both time points

(p<0.01), while it was lost for HSVUV from 12h (p<0.01) to 18h p.s. Furthermore, at 18h p.s.

HSVINF-induced activation of CD56bright

NK cells was significantly stronger than HSVUV-

induced activation of CD56bright

NK cells (p<0.05). The discrepancy in kinetics and activation

of CD56bright

NK cells between HSVINF and HSVUV indicates that viral infectivity might be

important for HSV-1-induced NK cell activation.

Results

36

FIG. 6. HSV-1 induces CD69 up-regulation on NK cells. PBMC were stimulated with CpG-A, UV-inactivated

(HSVUV) and infectious (HSVINF) HSV-1, and IL-2, or left unstimulated (mock) as control, and analyzed by flow

cytometry (FACS). NK cells were gated as CD56-positive CD3- and CD14-negative population and analyzed for

CD69 expression. A. Representative FACS plot of NK cells 12h and 18h post stimulation (p.s.) B and C. CD69-

expressing NK cells (%) 12h (B) and 18h (C) p.s. , given as mean and standard error of 15 (B) and seven (C)

independent experiments. ##

p<0.01 vs. mock; * p<0.05, ** p<0.01 as indicated (Tukey HSD).

Results

37

FIG. 7. NK cell sub-populations are activated differently. PBMC were stimulated with CpG-A,

UV-inactivated (HSVUV) and infectious (HSVINF) HSV-1, and IL-2, or left unstimulated (mock) as control, and

analyzed by flow cytometry (FACS). NK cell sub-populations were gated as CD56-low positive (CD56dim

) CD3-

and CD14-negative and CD56-high positive (CD56bright

) CD3- and CD14-negative populations and analyzed for

CD69 expression. A and B. CD69-expressing CD56dim

(A) and CD56bright

(B) NK cells (%) 12h post stimulation

(p.s.), given as mean and standard error of 15 independent experiments. C and D. CD69-expressing CD56dim

(C)

and CD56bright

(D) NK cells (%) 18h p.s. , given as mean and standard error of seven independent experiments. ##

p<0.01 vs. mock; ** p<0.01 as indicated (Tukey HSD).

Results

38

5.2 Only infectious HSV-1 induces NK cell effector functions

We next wanted to know, if CD69 up-regulation reflected induction of NK cell effector

functions, so we investigated NK cell IFN- secretion, using a Cytokine Secretion Assay

Detection Kit (Miltenyi Biotec), and NK cell degranulation, detecting CD107a surface

expression. CD107a, also called lysosomal-associated membrane protein-1 (LAMP-1), lines

the membranes of endosomal and secretory vesicles, like cytolytic granules, and is normally

not expressed on the outer cellular membrane. Upon release of cytolytic granules, CD107a is

temporarily present on the cell surface, therefore serving as an indicator of degranulation. The

correlation of CD107a surface expression with cytokine secretion and in particular

cytotoxicity has been demonstrated for NK cells (Alter et al., 2004). NK cell effector

functions were measured at 12h p.s. (FIG. 8A). CpG-A and HSVUV failed to induce NK cell

IFN- secretion as well as degranulation, whereas IL-2 stimulated IFN- secretion (p<0.05)

(FIG. 8B), but no degranulation (FIG. 8C). HSVINF induced significant IFN- secretion by NK

cells compared to the mock control (p<0.01), and also compared to CpG-A (p<0.01), HSVUV

(p<0.05) and IL-2 (p<0.05) (FIG. 8B). HSVINF was also the only stimulus leading to

significant degranulation compared to the mock control and all other stimuli (p<0.01)

(FIG. 8C). The fact that only HSVINF, not HSVUV, was able to induce NK cell effector

functions further supports the relevance of HSV-1 infectivity for full activation of NK cells

and their effector functions. Interestingly, HSVINF-induced effector functions were not

restricted to one of the NK cell subpopulations, as reported in the literature, namely IFN-

secretion mainly by CD56bright

cells and degranulation, indicating cytotoxicity, mostly by

CD56dim

cells (Cooper et al., 2001a). NK cell effector functions rather seemed evenly

distributed between both subpopulations (FIG. 8A), without significant differences in IFN-

secretion and degranulation between CD56bright

cells and CD56dim

cells (data not shown).

Results

39

FIG. 8. Infectious HSV-1 induces NK cell effector functions. PBMC were stimulated with CpG-A,

UV-inactivated (HSVUV) and infectious (HSVINF) HSV-1, and IL-2, or left unstimulated (mock) as control, and

analyzed by flow cytometry (FACS) 12h post stimulation (p.s.). NK cells were gated as CD56-positive CD3- and

CD14-negative population and analyzed for IFN- secretion and CD107a surface expression. A. Representative

FACS plot of NK cell IFN- secretion and CD107a surface expression. B and C. IFN--secreting (B) and

CD107a-expressing (C) NK cells (%), given as mean and standard error of 15 independent experiments.

# p<0.05,

## p<0.01 vs. mock; * p<0.05, ** p<0.01 as indicated (Tukey HSD).

Results

40

5.3 HSV-1 activates NK cells in part via type I IFN induction

Type I IFN have been published to be crucial in anti-HSV resistance and also of importance

in HSV-induced NK cell activation (Dupuis et al., 2003; Gill et al., 2011; Feldman et al.,

1992). PDC are known to be a major source of type I IFN (Siegal et al., 1999) and to secrete

high amounts upon stimulation with HSV-1 (Schuster et al., 2010). We therefore decided to

investigate HSV-1-induced IFN-2a/2b (IFN-) secretion within PBMC and by purified

PDC. CpG-A as well as HSVUV and HSVINF induced significant IFN- secretion compared to

the mock control at 12h (p<0.01) (FIG. 9A) and at 18h p.s. (p<0.01, p<0.05 and p<0.01,

respectively) (FIG. 9B). Kinetics of IFN- secretion appeared to be similar to kinetics of

CD69 up-regulation, in that HSVINF-induced IFN- secretion was significantly higher than

CpG-A-induced (p<0.01) and HSVUV-induced IFN- secretion (p<0.05) at 12h p.s. , with the

significant difference getting lost by 18h p.s. CpG-A actually was the strongest IFN--

inducing stimulus at 18h p.s.

FIG. 9. HSV-1 induces IFN- secretion within PBMC. PBMC were stimulated with CpG-A, UV-inactivated

(HSVUV) and infectious (HSVINF) HSV-1, and IL-2, or left unstimulated (mock) as control, and supernatants

were analyzed for IFN-2a/2b using enzyme-linked immunosorbent assay (ELISA). A and B. IFN-2a/2b

(IFN-) secretion within PBMC (pg/ml) 12h (A) and 18h (B) post stimulation (p.s.), given as mean and standard

error of 15 (A) and seven (B) independent experiments. #

p<0.05, ##

p<0.01 vs. mock; * p<0.05, ** p<0.01 as

indicated (Tukey HSD).

Results

41

Stimulation of 5x105 purified PDC with HSVINF led to a mean IFN- secretion into

supernatants of about 10ng/ml (data not shown), and we decided to examine the potential of

PDC-derived supernatants (PDC-SN) to stimulate CD69 up-regulation on purified NK cells.

Kinetic studies revealed a rapid effect of PDC-SN on NK cells. Supernatants containing an

INF-2a/2b concentration of 20pg/ml lead to significant CD69 up-regulation compared to the

mock control within 12h (p<0.05) and supernatants of 40 or 80pg/ml INF-2a2b did this even

within 3h (p<0.05 and p<0.01, respectively) (FIG 10A). This demonstrates that PDC-SN of

low IFN- concentrations are sufficient to activate NK cells within few hours. Interestingly,

NK cell CD69 up-regulation mediated by PDC-SN seemed to occur in two phases, namely

between 3 and 6h and between 12 and 18h p.s. , with a plateau between 6 and 12h p.s.

In order to figure out if NK cell activation induced by PDC-SN was due to IFN- or other

type I IFN, we compared PDC-SN with recombinant human IFN-2b (rhIFN-) containing

equal IFN-2a/2b concentrations and further used an antibody against the IFN-/ receptor

(IFN-R) to block any impact of type I IFN. Stimulation of purified NK cells with serial

dilutions of PDC-SN and rhIFN- confirmed IFN- as potent stimulus for NK cell activation,

with saturation beginning at IFN-2a/2b concentrations between 32 and 64pg/ml (FIG. 10B).

The effect of PDC-SN on CD69 up-regulation was more potent than the effect of rhIFN-.

Neutralization of IFN-R diminished CD69 up-regulation caused by PDC-SN significantly

(p<0.05), proving type I IFN as major soluble factors in PDC-dependent NK cell activation

after stimulation with HSV-1. CD69 up-regulation by rhIFN- was reduced clearly, but not

significantly (FIG. 10C).

Results

42

FIG. 10. PDC-dependent NK cell activation by HSV-1 is mediated by type I IFN. Purified NK cells were

stimulated with supernatants derived from purified HSV-1-stimulated PDC (PDC-SN), and human recombinant

IFN-2b (rhIFN-) or left unstimulated (mock) as control, and analyzed by flow cytometry (FACS) for CD69

expression (%). A. Kinetics of NK cell activation by PDC-SN containing different concentrations of IFN-2a/2b

(IFN-), given as mean and standard error of three independent experiments. B. Serial dilution of PDC-SN and

rhIFN-. CD69 expression 18h post stimulation (p.s.), given as mean and standard error of four independent

experiments. C. CD69 expression 18h p.s. in the presence of a neutralizing antibody against the IFN-/

receptor (IFN-R), given as mean and standard error of four independent experiments. # p<0.05,

## p<0.01 vs.

mock (Tukey HSD); * p≤0.05 as indicated (Student’s t-test).

Results

43

5.4 TNF- plays a major role in HSV-1-induced NK cell activation

PDC-SN seemed to be more potent in activating NK cells than rhIFN-, and within PBMC

more cell populations are present besides PDC, so other cytokines besides type I IFN could be

involved in HSV-1-induced NK cell activation. We therefore decided to check supernatants of

CpG-A- and HSV-1-stimulated PBMC for further cytokines using a Human Th1/Th2 11plex

bead array (Affymetrix eBioscience). Neither stimulus caused significant levels of IL-12,

IFN-, IL-2, IL-10, IL-4, IL-5 or TNF- within supernatants. Significant IL-6 secretion

compared to the mock control was induced by CpG-A (p<0.01) (data not shown), while IL-8

was secreted in all samples including the mock control (FIG. 11). Two cytokines, namely

IL-1 and TNF-, were significantly increased in HSVINF-stimulated PBMC compared to the

mock control and to HSVUV-stimulated PBMC (p<0.05). These findings suggest that IL-1

and TNF- might be involved in the stimulation of NK cell activation and in particular NK

cell effector functions by HSVINF.

FIG. 11. Infectious HSV-1 induces secretion of IL-1 and TNF-. PBMC were stimulated for 18h with

CpG-A, UV-inactivated (HSVUV) and infectious (HSVINF) HSV-1, or left unstimulated (mock) as control, and

supernatants were analyzed for cytokines using an 11plex bead array (Affymetrix eBioscience). Secretion of

IL-8, IL-1, and TNF- within PBMC (pg/ml), given as mean and standard error of seven independent

experiments. #

p<0.05 vs. mock; * p<0.05 as indicated (Tukey HSD).

Results

44

In order to investigate the effect of PDC-derived type I IFN, and also of IL-1 and TNF-,

more closely, neutralization experiments were conducted. PBMC were stimulated with

CpG-A, HSVUV and HSVINF in the presence of antibodies against IFN-R, IL-1 and TNF-

or the respective isotype controls. At 12h p.s. NK cell CD69 up-regulation, IFN- secretion

and degranulation as well as IFN- secretion within PBMC were determined. Neutralization

of TNF- significantly decreased CD69 up-regulation induced by CpG-A, HSVUV and

HSVINF (p<0.01) (FIG. 12A), and it also significantly reduced HSVINF-induced IFN-

secretion (p<0.05) (FIG. 12B), while it did not affect NK cell degranulation (FIG. 12C).

Blocking of IFN-R significantly diminished CpG-A- and HSVUV-induced CD69 up-

regulation (p=0.05 and p<0.01, respectively), but had only a minimal effect on HSVINF-

induced CD69 up-regulation (FIG. 12A) and no inhibitory effect on NK cell effector

functions (FIG. 12B, C). In fact, neutralization of the IFN-R even increased IFN- secretion,

although not significantly (FIG. 12B). In contrast, neutralization of IL-1 did neither

influence NK cell activation nor NK cell effector functions. IFN- secretion was reduced by

neutralization of IFN-R, in consistence with the known autokrine loop (Marie et al., 1998),

and interestingly, also by neutralization of TNF- as well as IL-1 (FIG. 12D). Reduction of

IFN- levels was distinct, although only significant for CpG-A (p<0.05 for TNF- and

IFN-R). Simultaneous neutralization of TNF- and IFN-R did not result in increased

effects on HSVINF-induced CD69 up-regulation (FIG. 13A), degranulation (FIG. 13C) or

IFN- secretion (FIG. 13D), while the increase of IFN- secretion observed after IFN-R

neutralization was abolished by combination of both antibodies (FIG. 13B). These findings

indicate a crucial role for TNF- in HSVINF-induced NK cell activation and IFN- secretion,

whereas it is negligible in HSVINF-induced NK cell degranulation. Besides, all three cytokines

seem to be required for the secretion of large amounts of IFN- upon stimulation with either

CpG-A or HSV-1. Furthermore, combined neutralization of TNF- and IFN-R suggests

opposed functions of TNF- and type I IFN in IFN- induction by HSVINF.

Results

45

FIG. 12. TNF- plays a major role in HSV-1-induced NK cell activation. PBMC were stimulated for 12h

with CpG-A, UV-inactivated (HSVUV) and infectious (HSVINF) HSV-1, or left unstimulated (mock) as control, in

the presence of neutralizing antibodies against IL-1 (IL-1) and TNF- (TNF-), and an isotype control

(IgG1), against the IFN-/ receptor (IFN-R), and an isotype control (IgG2a). Cells were analyzed by flow

cytometry (FACS), supernatants by enzyme-linked immunosorbent assay (ELISA). A - C. NK cells were gated

as CD56-positive CD3- and CD14-negative population and analyzed for CD69 expression (A), IFN- secretion

(B) and CD107a surface expression (C) (%). D. IFN-2a/2b (IFN-) secretion within PBMC (pg/ml). All values

are given as mean and standard error of five independent experiments. * p≤0.05, ** p≤0.01 as indicated

(Student’s t-test).

Results

46

FIG. 13. Simultaneous neutralization of TNF- and IFN-R does not increase the inhibitory effect of

TNF- neutralization. PBMC were stimulated for 12h with CpG-A, UV-inactivated (HSVUV) and infectious

(HSVINF) HSV-1, or left unstimulated (mock) as control, in the presence of neutralizing antibodies against

TNF- (TNF-), and an isotype control (IgG1), against the IFN-/ receptor (IFN-R), and an isotype

control (IgG2a) or both TNF- and IFN-R, and both isotype controls. Cells were analyzed by flow cytometry

(FACS), supernatants by enzyme-linked immunosorbent assay (ELISA). A - C. NK cells were gated as CD56-

positive CD3- and CD14-negative population and analyzed for CD69 expression (A), IFN- secretion (B) and

CD107a surface expression (C) (%). D. IFN-2a/2b (IFN-) secretion within PBMC (pg/ml). All values are

given as mean and standard error of three independent experiments.

Results

47

5.5 Monocytes contribute to HSV-1-induced TNF- production

Since TNF- seemed to be the key cytokine in HSVINF-induced NK cell activation within

PBMC, we were interested in which cell populations might be responsible for TNF-

production and performed a Cytokine Secretion Assay (Miltenyi Biotec) to detect

TNF--secreting cells within PBMC. We analyzed TNF- secretion within seven different

cell populations, namely PDC, monocytes, B cells, NK cells, T cells, CD4+ T cells and

CD8+ T cells, upon stimulation with CpG-A, HSVUV and HSVINF. We first looked at TNF-

secretion within the individual cell populations and could identify PDC and monocytes as

major TNF- sources with significant secretion upon stimulation with CpG-A (p<0.01),

HSVUV (p<0.01 for PDC and p<0.05 for monocytes) and HSVINF compared to mock (p<0.01)

(FIG. 14A). In addition, CpG-A stimulated TNF- secretion within the B cell population

(p<0.01), while HSVINF induced TNF- secretion within B cells (p<0.01), NK cells (p<0.01),

T cells (p<0.05), CD4+ T cells (p<0.01) and CD8

+ T cells (p<0.05). A significant

difference in TNF- secretion between HSVUV and HSVINF stimulation was observed within

monocytes (p<0.01). We next decided to identify total TNF- secretion within PBMC. For

this purpose we multiplied TNF- secretion within each cell population (FIG. 14A) with the

frequency of the respective cell population within PBMC (FIG. 14B), resulting in each cell

population’s TNF- secretion within PBMC, and combined TNF- secretion of all cell

populations, to get total TNF- secretion within PBMC (FIG. 14C). Interestingly, only

CpG-A and HSVINF induced significant overall TNF- secretion within PBMC compared to

the mock control (p<0.01), and HSVINF-induced TNF- secretion also differed significantly

from HSVUV-induced TNF- secretion (p<0.05), confirming the results of the bead array

(FIG. 11). Notably, monocytes appeared to be key producers of TNF- in this analysis

(FIG. 14C).

Results

48

FIG. 14. Monocytes contribute to HSV-1-induced TNF- production. PBMC were stimulated for 18h with

CpG-A, UV-inactivated (HSVUV) and infectious (HSVINF) HSV-1, or left unstimulated (mock) as control, and

analyzed by flow cytometry (FACS). PDC were gated as CD304-positive CD3- and CD14-negative, monocytes

as CD14-positive, B cells as CD19-positive CD3- and CD14-negative, NK cells as CD56-positive CD3- and

CD14-negative, T cells as CD3- and TCR-positive CD14-negative, CD4+ T cells as CD3- and CD4-positive

CD14-negative, and CD8+ T cells as CD3- and CD8-positive CD14-negative population. A. TNF--secreting

cells (%) within the respective individual cell population. B. Frequency of each cell population (%) within

PBMC. C. TNF--secreting cells (%) within PBMC. All values are given as mean (A - C) and standard error (A,

C) of five independent experiments. #

p<0.05, ##

p<0.01 vs. mock; * p<0.05, ** p<0.01 as indicated (Tukey

HSD).

Results

49

5.6 Monocytes can be infected by HSV-1

Next, we decided to engage in HSV-1 infection experiments, because NK cells are known to

recognize infected cells as target cells (Vivier, 2006). Monocyte activation seemed to be

particularly influenced by HSV-1 infectivity, and several working groups could already

demonstrate infection of mononuclear phagocytes (Daniels et al., 1978; Albers et al., 1989).

Since preliminary experiments of PBMC infected with a virus isolate (HSVGFP) expressing a

GFP-VP22 fusion protein also hinted at monocytes as HSV-1 target cells within PBMC (data

not shown), we conducted infection experiments with isolated monocytes. We noticed that

monocytes, which had been purified using magnetic beads specific for CD14, were only in

part positive for CD14 after being in cell culture for 24h or 48h (FIG. 15). Labeling of freshly

isolated monocytes evidenced a purity of about 95%, and staining of monocytes for lineage

markers as well as specific phagocyte markers demonstrated a contamination by other cells of

less than 5% after cultivation (data not shown). Thus, monocytes appear to down-regulate

CD14 when being cultured.

FIG. 15. Monocytes down-regulate CD14 upon cultivation. Monocytes were purified by magnetic-activated

cell sorting (MACS) using CD14-coupled beads and analyzed by flow cytometry (FACS) for CD14 expression.

Representative FACS plot of monocytes immediately post purification (p.p.) and 24h and 48h p.p.

Monocytes were infected with HSVGFP and analyzed for green fluorescence. HSVINF was used

as infectious non-fluorescent and HSVUV as non-infectious non-fluorescent control virus. In

fact, monocytes infected with HSVGFP exhibited significant green fluorescence compared to

the mock control and the two non-fluorescent viruses HSVUV and HSVINF at 24h (p<0.01) and

48h post infection (p.i.) (p<0.01 for mock and HSVUV, n.s. for HSVINF), demonstrating

infection of monocytes (FIG. 16A). The percentage of HSVGFP-infected monocytes declined

Results

50

from 24h to 48h p.i. , indicating rather abortive than productive HSV-1 infection, in

concordance with observations of several working groups (Daniels et al., 1978; Albers et al.,

1989; Bruun et al., 1998). In order to determine productivity of monocyte infection we

analyzed supernatants of HSV-1-infected monocytes and HSV-1-infected human foreskin

fibroblasts (HFF) as control cells for HSV-1 DNA, using quantitative PCR. Cells were

infected with HSVGFP and with another HSV-1 variant (HSVd106S), which is infectious, but

non-replicative (Liu et al., 2009). Infected cells were cultured for up to 5 days (FIG. 16B).

HSV-1 DNA increased over time in supernatants of HSVGFP-infected HFF, whereas it

declined in supernatants of HSVd106S-infected HFF, corresponding to the replication capacities

of HSVGFP and HSVd106S. In contrast, HSV-1 DNA dropped in supernatants of HSVd106S- as

well as HSVGFP-infected monocytes. These results confirm non-productive infection of

monocytes by HSV-1.

FIG. 16. Monocytes are non-productively infected by HSV-1. A. Purified monocytes were infected with

UV-inactivated (HSVUV), infectious (HSVINF), and infectious GFP-expressing (HSVGFP) HSV-1, or left

uninfected (mock) as control, and analyzed by flow cytometry (FACS) for green fluorescence (%) 24h and 48h

post infection (p.i.). Values are given as mean and standard error of eleven independent experiments. ##

p<0.01

vs. mock; ** p<0.01 as indicated (Tukey HSD). B. Purified monocytes of three different donors and human

foreskin fibroblasts (HFF) were infected with infectious GFP-expressing (HSVGFP) and an infectious but

replication-deficient GFP-expressing (HSVd106S) HSV-1 and cultivated for different time periods. Supernatants

of the indicated time points were analyzed by quantitative PCR for viral load (copies/ml). Values of monocytes

are given as mean of three different donors.

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51

5.7 Monocytes up-regulate MHC-I upon exposure to infectious HSV-1

Since HSV-1 has been published to down-regulate MHC-I molecules via ICP47 (Hill et al.,

1995; Früh et al., 1995), which might be responsible for HSVINF-induced NK cell activation

(Huard and Früh, 2000), we checked for expression of classical HLA-ABC and non-classical

HLA-E. Monocytes inoculated with HSVINF and HSVGFP exhibited significant HLA-ABC up-

regulation compared to the mock control at 24h and 48h p.i. (p<0.01), with rising kinetics

from 24h to 48h p.i. (p<0.01) (FIG. 17A). In contrast, HSVUV did not induce HLA-ABC up-

regulation, but behaved like the mock control with significant differences to both HSVINF and

HSVGFP at 24h and 48h p.i. (p<0.01). HLA-E was regulated in a similar manner to

HLA-ABC, with overall up-regulation being induced by HSVINF and HSVGFP (FIG. 17B).

Although plotting green fluorescence against HLA-ABC and HLA-E expression indicated

MHC-I down-regulation in few infected monocytes, overall up-regulation of MHC-I in

monocyte cultures was much more distinct (FIG. 18).

FIG. 17. Monocytes up-regulate MHC-I upon exposure to infectious HSV-1. Purified monocytes were

infected with UV-inactivated (HSVUV), infectious (HSVINF), and infectious GFP-expressing (HSVGFP) HSV-1, or

left uninfected (mock) as control, and analyzed by flow cytometry (FACS) for MHC-I expression (MFI) 24h and

48h post infection (p.i.). A. Fold change of HLA-ABC expression, given as mean and standard error of eleven

independent experiments. ##

p<0.01 vs. mock; ** p<0.01 as indicated (Tukey HSD). B. Fold change of HLA-E

expression, given as mean of two (HSVUV, HSVINF) and three (HSVGFP) independent experiments.

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52

FIG. 18. Monocytes are infected by HSV-1 and up-regulate MHC-I upon exposure to infectious HSV-1. Purified monocytes were infected with UV-inactivated (HSVUV), infectious (HSVINF), and infectious

GFP-expressing (HSVGFP) HSV-1, or left uninfected (mock) as control, and analyzed by flow cytometry (FACS)

for green fluorescence and MHC-I expression 24h and 48h post infection (p.i.). Representative FACS plot of

green fluorescence (GFP) and HLA-ABC and HLA-E expression.

Since type I IFN are known to induce up-regulation of MHC-I molecules (Samuel, 2001), we

checked monocyte supernatants for INF- and observed reproducible secretion only by

monocytes inoculated with HSVINF and HSVGFP (p<0.05 for HSVGFP vs. mock and vs.

HSVUV, at 24h and 48h p.i.), but not HSVUV (FIG. 19).

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FIG. 19. Infectious HSV-1 induces IFN- secretion by monocytes. Purified monocytes were infected with

UV-inactivated (HSVUV), infectious (HSVINF), and infectious GFP-expressing (HSVGFP) HSV-1, or left

uninfected (mock) as control, and supernatants were analyzed for IFN-2a/2b using enzyme-linked

immunosorbent assay (ELISA). IFN-2a/2b (IFN-) secretion at 24h and 48h post infection (p.i.), given as

mean and standard error of ten independent experiments. #

p<0.05 vs. mock; * p<0.05 as indicated (Tukey

HSD).

In order to test the hypothesis, that type I IFN were responsible for HLA-ABCE up-regulation

after HSV-1 infection, we performed neutralization experiments, where we infected

monocytes with HSVGFP in the presence of IFN-R and the isotype control (IgG2a).

Comparing IFN-R with IgG2a revealed distinct effects of type I IFN on monocyte infection

as well as HLA-ABCE regulation (FIG. 20).

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54

FIG. 20. Type I IFN suppress HSV-1 infection of monocytes and induce up-regulation of MHC-I. Purified

monocytes were infected with infectious GFP-expressing HSV-1 (HSVGFP) in the presence of a neutralizing

antibody against the IFN-/ receptor (IFN-R) and an isotype control (IgG2a) and analyzed by flow

cytometry (FACS) for green fluorescence and MHC-I expression 24h and 48h post infection (p.i.).

Representative FACS plot of green fluorescence (GFP) and HLA-ABC and HLA-E expression.

Neutralization of IFN-R increased monocyte infection at 24h (p=0.05) and 48h (p<0.05) p.i.

(FIG. 21A) and prevented up-regulation of HLA-ABC at 24h (n.s.) and 48h (p<0.05) p.i.

(FIG. 21B) as well as HLA-E at 24h (p<0.05) and 48h (p<0.01) p.i. (FIG. 21C). Furthermore,

IFN-R neutralization significantly diminished IFN- secretion at 24h and 48h p.i. (p<0.05)

(FIG. 21D), once again confirming the positive feedback loop for IFN- production (Marie et

al., 1998) (FIG. 12D). These results prove type I IFN as cause of HSV-1-induced HLA-ABCE

up-regulation by monocytes and further propose type I IFN as potential restriction factors for

productive HSV-1 infection and replication in monocytes.

Results

55

FIG. 21. Type I IFN suppress HSV-1 infection of monocytes, induce up-regulation of MHC-I and trigger

IFN- secretion. Purified monocytes were infected with infectious GFP-expressing HSV-1 (HSVGFP) in the

presence of a neutralizing antibody against the IFN-/ receptor (IFN-R) and an isotype control (IgG2a), or

left uninfected (mock) as control, and analyzed by flow cytometry (FACS) 24h and 48h post infection (p.i.),

supernatants were analyzed using enzyme-linked immunosorbent assay (ELISA). A. Green fluorescent

monocytes (%). B. Fold change of HLA-ABC expression (MFI). C. Fold change of HLA-E expression (MFI). D.

IFN-2a/2b (IFN-) secretion. All values are given as mean and standard error of three independent

experiments. * p≤0.05, ** p≤0.01 as indicated (Student’s t-test).

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56

5.8 HSVd106S affects monocytes similar to HSVGFP

GFP is coupled to the tegument protein VP22 in HSVGFP and is thus present within viral

particles, so HSVGFP particles themselves fluoresce. Consequently, green fluorescing

monocytes may not be infected monocytes expressing GFP, but monocytes with fluorescing

viral particles sticking to them. We therefore repeated our infection experiments with

HSVd106S, which carries the GFP gene under the control of a human cytomegalovirus

(HCMV) promoter. HSVd106S particles do not fluoresce, so monocytes can only fluoresce

when they have been infected and express GFP. Infection of monocytes with HSVd106S had

effects similar to infection with HSVGFP. HSVd106S induced significant fluorescence at 24h p.i.

(p<0.05) (FIG. 22A), that was lost at 48h p.i. HSVd106S infection induced up-regulation of

HLA-ABC at 24h and 48h p.i. (p<0.01) (FIG. 22B) and of HLA-E at 24h (p<0.05) and 48h

p.i. (FIG. 22C). HSVd106S-infected monocytes secreted even higher amounts of IFN- than

HSVGFP-infected monocytes (FIG. 22D). Similarities in fluorescence as well as IFN-

induction and HLA-ABCE up-regulation induced by both viruses argue for an actual infection

of monocytes by HSV-1.

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57

FIG. 22. HSVd106S affects monocytes similar to HSVGFP. Purified monocytes were infected with infectious

GFP-expressing (HSVGFP) and an infectious but replication-deficient GFP-expressing (HSVd106S) HSV-1, or left

uninfected (mock) as control, and analyzed by flow cytometry (FACS) 24h and 48h post infection (p.i.),

supernatants were analyzed using enzyme-linked immunosorbent assay (ELISA). A. Green fluorescent

monocytes (%). B. Fold change of HLA-ABC expression (MFI). C. Fold change of HLA-E expression (MFI). D.

IFN-2a/2b (IFN-) secretion. All values are given as mean and standard error of six (A, D), five (B), and three

(C) independent experiments. # p≤0.05,

## p≤0.01 HSVd106S vs. mock (Student’s t-test).

Results

58

Altogether, infection experiments demonstrated monocytes as target cells for HSVINF,

suggesting them as crucial cell population in HSVINF-induced NK cell activation not only via

TNF- secretion, but via recognition of infected monocytes by NK cells. MHC-I down-

regulation by infected monocytes is a possible mechanism, yet according to our studies

unlikely. Furthermore, up-regulation of MHC class I polypeptide-related sequence (MIC) A

or B, which would be recognized by activating NK cell receptors (Vivier, 2006), could be

excluded in preliminary experiments (FIG. 23).

FIG. 23. HSV-1 does not induce up-regulation of MHC class I polypeptide-related sequence (MIC)A or B.

Purified monocytes were infected with infectious GFP-expressing HSV-1 (HSVGFP), or left uninfected (mock) as

control, and analyzed by flow cytometry (FACS) for MICA/B expression 24h post infection (p.i.).

Representative FACS plot of MICA/B expression.

Results

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5.9 Monocytes mediate NK cell effector functions upon HSV-1

infection within the PBMC context

In order to investigate the actual contribution of monocytes and also of PDC to

HSVINF-induced NK cell activation within the PBMC context, we conducted cell depletion

experiments, comparing non-depleted PBMC with monocyte- or PDC-depleted PBMC.

Depletion of monocytes as well as PDC decreased CD69 up-regulation, although to a variable

extent (FIG. 24A): CpG-A-induced CD69 up-regulation was significantly reduced only by

monocyte depletion (p<0.05), while it was diminished by both monocyte and PDC depletion

in the case of HSVUV (p<0.01) and HSVINF (p<0.01 and p<0.05, respectively). For HSVINF

stimulation the inhibitory effect of monocyte depletion was significantly stronger than the

effect of PDC depletion (p<0.01), which argues for monocytes to be more important in NK

cell activation than PDC when HSV-1 is infectious. HSVINF-induced NK cell effector

functions were both affected by cell depletion in the same manner. While depletion of PDC

had no effect on either effector function, depletion of monocytes prevented both IFN-

secretion (FIG. 24B) and degranulation (FIG. 24C). These results confirm PDC as important

cell population in NK cell activation by HSV-1, and they furthermore reveal monocytes as

key accessory cells in HSVINF-caused NK cell activation, and as indispensable cell population

for the induction of NK cell effector functions within the PBMC context. Interestingly, both

cell populations seem to be crucial for CpG-A- as well as HSV-1-stimulated IFN-

production (FIG. 24D). Depletion of monocytes as well as PDC reduced secretion of IFN-

induced by CpG-A, HSVUV (p<0.01 for monocyte depletion and p<0.05 for PDC depletion)

and HSVINF (p<0.01).

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FIG. 24. Monocytes mediate NK cell effector functions upon HSV-1 infection within the PBMC context.

PBMC were left non-depleted (PBMC) or were depleted of monocytes (PBMC monocytes) or of PDC

(PBMC PDC) and stimulated for 12h with CpG-A, UV-inactivated (HSVUV) and infectious (HSVINF) HSV-1,

or left unstimulated (mock) as control. Cells were analyzed by flow cytometry (FACS), supernatants by enzyme-

linked immunosorbent assay (ELISA). A - C. NK cells were gated as CD56-positive CD3- and CD14-negative

population and analyzed for CD69 expression (A), IFN- secretion (B) and CD107a surface expression (C) (%).

D. IFN-2a/2b (IFN-) secretion within PBMC (pg/ml). All values are given as mean and standard error of eight

independent experiments. * p<0.05, ** p<0.01 as indicated (Tukey HSD).

Results

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5.10 PDC serve as crucial accessory cell population in NK cell

activation by HSV-1-infected HFF

Donaghy et al. demonstrated the presence of PDC within recurrent genital herpes lesions and

their co-localization with NK cells (Donaghy et al., 2009), so we wanted to investigate the

role of PDC as accessory cell population in NK cell activation within infected tissue. In order

to simulate the situation of NK cell activation within tissue, we conducted experiments, in

which we inoculated NK cells with HSVUV and HSVINF and co-cultivated them with human

foreskin fibroblasts (HFF) in the absence and in the presence of PDC. NK cell-PDC ratios in

assays were adjusted to their physiological ratio within PBMC of the respective donors. At

24h p.s. , CD69 up-regulation on NK cells was measured. Clearly, HSV-1 does not activate

NK cells in a direct manner, since NK cells did not up-regulate CD69 in response to either

HSVUV or HSVINF, when cultured alone (FIG. 25A). Stimulation of NK cells with HSVUV and

in particular HSVINF in the presence of PDC led to moderate but not significant CD69

up-regulation. NK cells stimulated with HSV-infected HFF alone also slightly up-regulated

CD69, however, CD69 up-regulation was not significant. In contrast, when co-cultivated with

both HFF and PDC, NK cells stimulated with HSVUV as well as HSVINF significantly up-

regulated CD69 compared to the mock control (p<0.01). Checking IFN- levels, we observed

that PDC secreted considerably more IFN- in the presence of HFF (FIG. 25B). These results

indicate PDC as important accessory cells for NK cell activation within HSV-1-infected

tissue, possibly via secretion of type I IFN, and further suggest a dependence of PDC on a

sufficient cell density, and hence possible interactions with other cells, within the cell culture

to secrete high amounts of IFN- in response to HSV-1 stimulation, as observed by

Rönnblom et al. (Rönnblom et al., 1988).

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FIG. 25. PDC serve as crucial accessory cell population in NK cell activation by HSV-1-infected HFF.

Purified NK cells were cultivated alone or together with purified PDC, with HFF, or with HFF and PDC, and

stimulated for 24h with UV-inactivated (HSVUV) and infectious (HSVINF) HSV-1, or left unstimulated (mock) as

control. Cells were analyzed by flow cytometry (FACS), supernatants by enzyme-linked immunosorbent assay

(ELISA). A. NK cells were gated as CD56-positive CD3- and CD14-negative population and analyzed for CD69

expression (%). B. IFN-2a/2b (IFN-) secretion within cell culture (pg/ml). All values are given as mean and

standard error of three independent experiments. ##

p<0.01 vs. mock (Tukey HSD).

Interestingly, NK cells co-cultivated with HSV-infected HFF expressed less CD56 than

HSV-stimulated NK cells cultured without HFF. The decrease in CD56 expression was even

more obvious, when PDC were present (FIG. 26A). This effect was caused only by HSVINF,

not by HSVUV (FIG. 26B). CD56 expression on NK cells stimulated with HSVINF was

significantly lower compared to mock (p<0.05) and HSVUV (p<0.05). Apparently, HSV

infection of and / or replication within HFF induces NK cells to down-regulate CD56, the

effect being boosted by PDC.

Results

63

FIG. 26. NK cells co-cultivated with HSV-1-infected HFF down-regulate CD56. Purified NK cells were

cultivated alone or together with purified PDC, with HFF, or with HFF and PDC, and stimulated for 24h with

UV-inactivated (HSVUV) and infectious (HSVINF) HSV-1, or left unstimulated (mock) as control. NK cells were

gated as CD56-positive CD3- and CD14-negative population and analyzed by flow cytometry (FACS) for CD56

expression (MFI). A. Representative FACS plot of CD56 expression on NK cells after stimulation with HSVINF.

B. Fold change of CD56 expression, given as mean and standard error of three independent experiments.

# p<0.05 vs. mock; * p<0.05 as indicated (Tukey HSD).

Results

64

5.11 PDC supernatants inhibit HSV-1 replication in HFF

Type I IFN are known to lead to an antiviral state of virus-infected and -susceptible cells

(ISAACS and LINDENMANN, 1957). Since PDC are key producers of type I IFN (Siegal et

al., 1999; Cella et al., 1999) and furthermore have been shown to suppress HSV-2 replication

upon vaginal infection (Lund et al., 2006), we decided to examine the potential of PDC-SN to

inhibit HSV-1 replication in HFF. For this purpose, HFF were infected with HSVGFP at an

MOI of 0.001 and 0.01 and cultivated for 24h and 48h in the absence and presence of

PDC-SN corresponding to IFN-2a/2b levels of 20pg/ml and 200pg/ml. Infection rates were

determined via GFP expression in HFF. The first observation we made was the wide range of

infection rates in HFF cultured without PDC-SN (FIG. 28A), varying for MOI 0.001 between

0.1% and 2.6% at 24h and between 0.5% and 82.0% at 48h, for MOI 0.01 between 0.1% and

19.5% at 24h and between 19.3% and 99.4% at 48h p.i. Obviously, productivity of HSV-1

replication depends on the current state and condition of the infected cell, which seems to be

variable for HFF. However, when infected HFF were cultured in the presence of PDC-SN,

infection rates were reduced compared to infection rates in the absence of PDC-SN (FIG. 27).

Relative reduction of infection rates was significant at 24h p.i. for MOI 0.01 at 20pg/ml and

200pg/ml (p<0.05) (FIG. 28B), at 48h p.i. for MOI 0.001 at 200pg/ml (p<0.05) and for MOI

0.01 at 20pg/ml and 200pg/ml (p<0.01) (FIG. 28C). These results evidence the potential of

PDC to directly inhibit HSV-1 replication in target cells via secretion of antiviral cytokines,

most likely type I IFN.

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65

FIG. 27. HSV-1 replication in HFF decreases in the presence of PDC supernatants. HFF were infected with

infectious GFP-expressing HSV-1 (HSVGFP) at a MOI of 0.001 and a MOI of 0.01, in the absence of PDC

supernatants (w/o PDC-SN) and in the presence of PDC supernatants containing 20pg/ml and 200pg/ml

IFN-2a/2b (IFN-), or left uninfected (mock) as control, and analyzed by flow cytometry (FACS) for green

fluorescence 24h and 48h post infection (p.i.). Representative FACS plot of green fluorescence (GFP).

Results

66

FIG. 28. PDC supernatants inhibit HSV-1 replication in HFF. HFF were infected with infectious

GFP-expressing HSV-1 (HSVGFP) at a MOI of 0.001 and a MOI of 0.01, in the absence of PDC supernatants

(w/o PDC-SN) and in the presence of PDC supernatants containing 20pg/ml and 200pg/ml IFN-2a/2b (IFN-),

or left uninfected (mock) as control, and analyzed by flow cytometry (FACS) for green fluorescence 24h and

48h post infection (p.i.). A. HSV-1 replication in HFF w/o PDC-SN, shown as green fluorescent HFF (%) 24h

and 48h p.i. B and C. HSV-1 replication in the presence of PDC-SN, shown as fold change of green fluorescent

HFF (%) compared to infection w/o PDC-SN 24h (B) and 48h (C) p.i. All values are given as mean and standard

error of three independent experiments. #

p<0.05, ##

p<0.01 vs. w/o PDC-SN (Tukey HSD).

Results

67

5.12 PDC-NK cell interactions are hampered in an HIV-1-infected

woman suffering from persisting genital ulcers

Human immunodeficiency virus type 1 (HIV-1) infection leads to a decrease in numbers as

well as function of PDC, leading to reduced IFN- secretion (Feldman et al., 2001; Schmidt

et al., 2005; Schmidt et al., 2006). It furthermore causes a defective crosstalk between PDC

and NK cells via functional defects of PDC and also NK cells (Reitano et al., 2009), on which

antiretroviral therapy has only minimal effects (Benlahrech et al., 2011). We therefore

analyzed PDC and NK cell activation in PBMC of an African woman infected with HIV-1

and suffering from immune reconstitution inflammatory syndrome (IRIS). Three months after

viral load decline and CD4+ T cell increase due to successful antiretroviral treatment she

developed painful genital ulcers due to HSV-2, which were only temporarily resolved by

several courses of aciclovir, topical application of imiquimod and a radical bilateral

vulvectomy (Strehl et al., 2012). Repeated virological analysis of the hyperproliferative

lesions revealed human papilloma virus type 54 (HPV-54) infection in addition to HSV-2

infection.

PBMC of this patient were stimulated with CpG-A, a TLR-7 agonist (S-27609), HSVUV and

HSVINF and analyzed at 18h p.s. for IFN- secretion, expression of markers for PDC

migration (CCR7), activation (CD80) and maturation (CD83) as well as NK cell activation

(CD69). Up-regulation of CCR7, CD80 and CD83 on stimulated but also on mock-cultivated

PDC suggested pre-stimulation of PDC in vivo (FIG. 29A). Activation of the patient’s NK

cells upon stimulation was severely impaired (FIG. 29B), as well as IFN- secretion within

PBMC (FIG. 29C), compared to a healthy control donor. These results suggest that impaired

IFN- production by PDC and subsequently reduced activation of NK cells contributed to the

patient’s disease.

Silencing of peripheral IFN- responses in HIV-1 infection has been associated with

enhanced interaction of CD40 on PDC with CD40 ligand (CD40L), a co-stimulatory

molecule, which is up-regulated upon immune activation (Donhauser et al., 2012). CD40L

levels transiently increase with the CD4+ T cell recovery upon antiretroviral therapy, which

might boost IFN-susceptible opportunistic infections in IRIS. Therefore, we retrospectively

analyzed levels of soluble (s)CD40L in the plasma of our patient (P1) and four other patients

Results

68

suffering from opportunistic infections (P2 - P5). Indeed, sCD40L levels in eight consecutive

plasma samples of P1 after initiation of antiretroviral treatment were significantly higher than

in cross-sectional samples of 52 untreated HIV-1-infected patients (p<0.001) (Donhauser et

al., 2012), and they were also higher than in the samples of P2 - P5 (P<0.01) (FIG. 29D).

Thus, suppression of TLR-7- and TLR-9-induced IFN- production by elevated sCD40L

levels may have contributed to the unusual and treatment-refractory genital ulcers in P1.

Alternatively, enhanced sCD40L levels may reflect prior in vivo stimulation caused by HSV-2

and HPV-54 infections. Altogether, these data indicate important interactions of PDC and NK

cells, which are hampered in immunosuppressed individuals and thus may lead to inefficient

control of persistent viral infections, such as human papilloma virus and herpes simplex virus

infections.

Results

69

FIG. 29. PDC-NK cell interactions are hampered in an HIV-1-infected woman suffering from persisting

genital ulcers. A. Surface marker expression on PDC of the investigated patient. PBMC were stimulated for 18h

with CpG-A, S-27609, UV-inactivated (HSVUV) and infectious (HSVINF) HSV-1, or left unstimulated (mock) as

control. Cells were analyzed by flow cytometry (FACS) for marker expression (%) indicating PDC migration

(CCR7), activation (CD80) and maturation (CD83) and compared to freshly isolated PBMC (baseline). B and C.

PBMC of the patient and a healthy control donor (control) were stimulated for 18h with CpG-A, S-27609,

UV-inactivated (HSVUV) and infectious (HSVINF) HSV-1, or left unstimulated (mock) as control. NK cells were

gated as CD56-positive CD3- and CD14-negative population and analyzed for CD69 expression (%) by flow

cytometry (FACS) (B), supernatants were analyzed for IFN-2a/2b (IFN-) secretion within PBMC (pg/ml) by

enzyme-linked immunosorbent assay (ELISA) (C). D. Plasma samples of the investigated patient (P1) and four

other IRIS cases (P2 - P5) were analyzed for sCD40L levels (ng/ml) by enzyme-linked immunosorbent assay

(ELISA). ** p<0.01 P2 - P5 vs. P1 (Tukey HSD).

Discussion

70

6 Discussion

Our analysis of human NK cell activation and induction of effector functions by different

stimuli revealed HSV-1 as potent and fast inducer of NK cell activation within the PBMC

context. Interestingly, we observed several differences concerning the stimulating potential

between HSVINF and HSVUV, first, on the level of NK cell activation and second, on the level

of cytokine secretion within PBMC (FIG. 30).

HSVINF induced overall NK cell activation significantly faster than HSVUV (FIG. 6B) and also

caused significantly stronger activation within the CD56bright

NK cell subset (FIG. 7D). In

addition, only HSVINF, and not HSVUV, induced significant NK cell IFN- secretion (FIG. 8B)

and degranulation (FIG. 8C). These results support the finding of Fitzgerald-Bocarsly and

colleagues that HSV-1-inoculated HFF were only lysed by human PBMC when the virus used

was infectious (Fitzgerald-Bocarsly et al., 1991), but stand in contrast to another study by

Ahmad et al., in which NK activity of HSV-1-stimulated human PBMC was similar for

infectious and UV-inactivated virus (Ahmad et al., 2000). This discrepancy could be caused

by the use of different methods. Ahmad et al. investigated the increase of basic lytic activity

against the NK cell target K562, whereas Fitzgerald-Bocarsly et al. analyzed lysis of HFF,

and we detected induction of degranulation in the absence of cytotoxicity-inducing target

cells.

Although both HSVINF and HSVUV caused secretion of exceedingly high amounts of IFN-

within PBMC, HSVINF-induced IFN- secretion was significantly faster and slightly stronger

than HSVUV-induced IFN- secretion (FIG. 9). Furthermore, in our study HSVINF stimulated

significant secretion of IL-1 and TNF-, while HSVUV failed to induce these two cytokines

within PBMC (FIG. 11), contradicting an early study published by Gosselin et al., who saw

similar TNF- induction in human PBMC by infectious and UV-irradiated HSV-1 (Gosselin

et al., 1992). Our observations indicate an effect of viral infectivity on the induction of pro-

inflammatory cytokines as well as on NK cell activation within the PBMC context and

suggest the necessity for viral infectivity in the induction of NK cell effector functions

(FIG. 30), either via induction of different cytokines, or via direct recognition of virus-

infected cells by NK cells as targets, or both.

Discussion

71

FIG. 30. Infectious and UV-inactivated HSV-1 exhibit different stimulation potentials within the PBMC

context. Both infectious and UV-inactivated HSV-1 are able to induce IFN-2a/2b (IFN-) secretion within

PBMC and CD69 up-regulation on NK cells, whereas only infectious HSV-1 causes secretion of TNF- and

IL-1 within PBMC and NK cell effector functions IFN- secretion and degranulation indicated by CD107a

surface expression. These findings suggest the importance of viral infectivity in complete activation of effector

NK cells by HSV-1.

NK cell effector functions were evenly distributed between the two NK cell subsets

(FIG. 8A), contradicting a previous concept of strict classification of CD56dim

and CD56bright

NK cells into a mainly cytotoxic and a major cytokine secreting subset, respectively (Cooper

et al., 2001a). However, Vivier proposed to rather define the CD56dim

and CD56bright

NK cell

subsets as “target cell responsive” and “cytokine responsive”, respectively, both possessing

the ability for cytotoxicity as well as cytokine secretion, depending on the stimulus (Vivier,

2006). Recently, De Maria et al. described CD56dim

NK cells as rapid producers of IFN-

upon antibody-mediated stimulation of natural killer receptors (De et al., 2011). We report

Discussion

72

here for the first time that infectious HSV-1 is a potent stimulus for IFN- secretion by

CD56dim

NK cells within the PBMC context.

INF- has been published to be of importance in HSV-induced NK cell activation (Gill et al.,

2011; Feldman et al., 1992) and several groups identified it as main cytokine in the induction

of NK cell activation after stimulation of human PDC with influenza virus, CpG, and poly

(I:C) (Benlahrech et al., 2009; Gerosa et al., 2005; Marshall et al., 2006; Romagnani et al.,

2005). Stimulation of purified NK cells with supernatants of HSVINF-stimulated PDC

(PDC-SN) demonstrated time- and dose-dependent induction of CD69 on NK cells by

PDC-SN (FIG. 10A). Interestingly, NK cell activation occurred in two phases, suggesting that

the initial and subsequent CD69 up-regulation were induced by two different mechanisms.

Further studies are required to identify the underlying mechanisms. The fact that

neutralization of the IFN-/ receptor significantly decreased PDC-SN-induced NK cell

activation (FIG. 10C) proves type I IFN as key cytokines in PDC-induced NK cell activation

after stimulation with HSV-1 (FIG. 31), consistent with PDC-induced NK cell activation after

stimulation with CpG-A (Benlahrech et al., 2009; Gerosa et al., 2005; Marshall et al., 2006;

Romagnani et al., 2005). However, NK cell activation by PDC-SN was slightly stronger than

NK cell activation by rhIFN- (FIG. 10B), and other groups described the involvement of

further cytokines, in particular TNF-, which collaborated with IFN- in PDC-induced NK

cell activation (Gerosa et al., 2005; Marshall et al., 2006; Romagnani et al., 2005). Actually,

we detected TNF- not only in PDC supernatants (data not shown), but also in PBMC

supernatants (FIG. 11).

Discussion

73

FIG. 31. PDC-dependent NK cell activation by HSV-1 is mediated by type I IFN. HSV-1-stimulated PDC

secrete high amounts of IFN-2a/2b (IFN-) and other type I IFN. Stimulation of purified NK cells with PDC

supernatants leads to CD69 up-regulation which is inhibited by a neutralizing antibody against the IFN-/

receptor. This identifies type I IFN as key cytokines in PDC-dependent NK cell activation by HSV-1.

TNF- has been shown to play an essential role in HSV infection in vivo. TNF- knockout

mice exhibited decreased survival rates in acute corneal HSV-1 infections and increased

reactivation rates after UV light stimulation (Minami et al., 2002), and lethal encephalitis after

intranasal HSV-1 infection (Sergerie et al., 2007). In our studies we demonstrated TNF- as

critical cytokine for CpG-A- and HSV-1-induced human NK cell activation (FIG. 12A) and

also for HSVINF-caused IFN- secretion (FIG. 12B) within the PBMC context. Cooper et al.

described the ability of IL-1 to co-stimulate IFN- production of CD56bright

NK cells together

with IL-12 or, in particular, IL-15 (Cooper et al., 2001b), however, we did not detect any

direct influence of IL-1 on NK cell activation or induction of effector functions

(FIG. 12A, B, C).

Type I IFN, which played a major role in PDC-mediated NK cell activation (FIG. 10),

appeared to be less important within the PBMC context. IFN-R neutralization only

significantly inhibited NK cell activation induced by CpG-A and HSVUV, but not by HSVINF

(FIG. 12A), and had no influence on degranulation (FIG. 12C). On the contrary, HSVINF-

induced IFN- secretion was increased by IFN-R neutralization (FIG. 12B), suggesting a

rather inhibitory influence of high amounts of type I IFN on NK cell IFN- secretion.

Similarly, Cousens et al. observed inhibition of murine IL-12 and subsequently IFN-

Discussion

74

secretion by type I IFN upon viral infection and bacterial stimulation (Cousens et al., 1997).

IFN- secretion after simultaneous neutralization of TNF- and IFN-R (FIG. 13B) points to

opposed functions of TNF- and type I IFN in IFN- induction by HSVINF, namely that

TNF- induces, and type I IFN rather inhibit HSV-1-induced IFN- production by NK cells.

We could observe a strict dependence of IFN- levels on type I IFN upon stimulation of

PBMC with GpG-A and HSV-1 (FIG. 12D) and also upon infection of monocytes with

HSV-1 (FIG. 21D), which is in concordance with the already published autocrine loop (Marie

et al., 1998). Interestingly, TNF- and IL-1 also strongly influenced IFN- secretion

(FIG. 12D). This suggests that secretion of high amounts of IFN- demands a positive

feedback loop consisting of a tight crosstalk of IFN--producing cells with each other and / or

other immune cells via production of type I IFN, TNF-, and IL-1 (FIG 32). Actually,

TNF- has been shown to induce secretion of low amounts of type I IFN, particularly IFN-,

in both human and mouse macrophages (Yarilina et al., 2008). Jimbo et al. demonstrated

IL-1 to be involved in a positive feedback loop increasing its own secretion by intervertebral

disc cells and also secretion of other inflammatory mediators like IL-6 and cyclooxygenase

(COX)-2 (Jimbo et al., 2005).

Discussion

75

FIG. 32. Secretion of high amounts of IFN-2a/2b (IFN-) within PBMC demands a positive feedback

loop involving type I IFN, TNF- and IL-1. HSV-1-induced secretion of IFN- is greatly diminished by

neutralization of the IFN-/ receptor as well as TNF- and IL-1, suggesting that not only type I IFN, but also

TNF- and IL-1 are involved in a positive feedback loop leading to secretion of high IFN-2a/2b (IFN-)

amounts after HSV-1 simulation of PBMC.

TNF- secretion assays revealed PDC and monocytes as potent TNF- sources upon

stimulation with HSVINF (FIG. 14A). Considering the much higher frequency of monocytes

within PBMC (FIG. 14B), monocytes have to be regarded as the most numerous TNF-

producers in the blood. Obviously, TNF- secretion by PDC did not depend on viral

infectivity, in contrast to monocytic TNF- secretion, which was significantly increased by

viral infectivity (FIG. 14A). Interestingly, with 16% the percentage of TNF--secreting

monocytes (FIG. 14A) was clearly higher than the percentage of infected monocytes of 2%

(FIG. 16A), which argues against TNF- secretion mainly by infected monocytes. It rather

suggests that infection of few monocytes stimulates a number of uninfected bystander

monocytes to secrete TNF-. The difference in the induction of TNF- secretion that we

observed between HSVINF and HSVUV on the PBMC level (FIG. 14C) could explain why

HSVUV was unable to mediate significant IFN- secretion by NK cells; neutralization of

TNF- significantly diminished NK cell IFN- secretion induced by HSVINF (FIG. 12B).

Discussion

76

These observations indicate that induction of certain NK cell effector functions, like IFN-

secretion, requires the secretion of pro-inflammatory cytokines within PBMC, which in turn

depends on viral infectivity. Induction of other NK cell effector functions, like cytotoxicity

indicated by degranulation, is independent at least from the cytokines investigated in our

study, but also depends on viral infectivity.

Ahmad et al. reported IL-15 as crucial cytokine in HSV-1-induced NK activity of human

PBMC, but in their study, infectivity of HSV-1 was not required for the induction of NK

activity (Ahmad et al., 2000), and in a later study Ahmad et al. showed that HSV-1-induced

up-regulation of IL-15 gene expression in monocytic cells was independent of viral infectivity

(Ahmad et al., 2007). We tested PBMC supernatants for secreted IL-15, but did not detect any

after HSV-1 stimulation (data not shown). Furthermore, the observations in the two other

studies would preclude IL-15 as critical factor for NK cell effector functions, since in our

studies only HSVINF induced NK cell effector functions, whereas HSVUV failed to induce

them (FIG 8). IL-18, which we did not investigate, might play a role in HSVINF-induced NK

cell activation and effector functions, since it has already been demonstrated to contribute to

NK cell activation in HSV-1-infected mice (Barr et al., 2007; Reading et al., 2007).

Infection experiments evidenced monocytes as target cells for HSV-1 which are infected, yet

to a very small percentage and without allowing productive viral replication (FIG. 16)

(FIG. 33), in concordance to prior studies of monocyte infection by HSV-1 (Bruun et al.,

1998; Daniels et al., 1978). Ineffective infection of and replication in monocytes was in part

due to monocytic type I IFN secretion, since IFN-R blocking significantly increased

infection rates in purified cells, in particular 48h p.i. (FIG. 21A).

Similarly, type I IFN were published to suppress HSV-1 replication in vitro in Vero cells,

HEp-2 cells, and fibroblasts (Härle et al., 2001; Noisakran et al., 2000) and to play a crucial

role in resistance to HSV infections in vivo (Dupuis et al., 2003; Casrouge et al., 2006).

Another restriction factor for HSV-1 replication in monocytes might be SAM domain and HD

domain-containing protein 1 (SAMHD1), which was recently shown to inhibit HSV-1

replication in differentiated macrophage cell lines (Kim et al., 2013).

Interestingly, IFN- secretion by monocytes depended on HSV-1 infectivity (FIG. 19), in

concordance to the observation of Melchjorsen et al., that cytokine induction by HSV in

Discussion

77

human monocyte-derived cells is dependent on virus replication (Melchjorsen et al., 2006).

This stands in contrast to IFN- secretion by PDC, which is induced by HSVINF as well as

HSVUV (Schuster et al., 2010) (FIG. 9). This difference is probably due to the fact that HSV-1

is able to infect monocytes (FIG. 16A) (Bruun et al., 1998; Daniels et al., 1978), but not PDC

(Schuster et al., 2010), and due to different recognition molecules involved; TLR-9 is

responsible for HSV-1 recognition in PDC (Krug et al., 2004), but not in monocytes, where

melanoma differentiation-associated protein 5 (MDA5) was shown to be the primary mediator

of HSV-1 recognition in macrophages (Melchjorsen et al., 2010). MDA5, a retinoic acid-

inducible gene (RIG)-I-related protein, senses viral RNA with a helicase domain and mediates

the induction of an antiviral response within the infected cell (Yoneyama et al., 2005). Since

the presence of herpesviral RNA requires the initiation of viral replication and therefore viral

infectivity, UV-inactivated HSV-1 should not be recognized by MDA5, explaining the lack of

IFN- induction in monocytes by HSVUV (FIG. 19). Interestingly, HSVd106S induced stronger

IFN- secretion than both infectious wildtype isolates (FIG. 22D). This might be due to a

deletion within the ICP27 gene of HSVd106S (Liu et al., 2009). Melchjorsen et al. determined

ICP27 as a factor counteracting cytokine induction in monocyte-derived cells by HSV

(Melchjorsen et al., 2006).

Discussion

78

FIG. 33. Monocytes are non-productively infected by HSV-1 and up-regulate MHC-I in a type I IFN-

dependent manner. HSV-1 is able to infect monocytes, but without production of new viral particles. HSV-1-

infected monocytes secrete low amounts of IFN-2a/2b (IFN-). Neutralization of the IFN-/ receptor

prevents MHC-I up-regulation induced by infectious HSV-1 (HSVINF) and increases infection rates in

monocytes. These data demonstrate non-productive infection of monocytes by HSV-1 leading to type I IFN-

dependent up-regulation of MHC-I. Furthermore, type I IFN prove to be involved in preventing productive

HSV-1 infection of and replication in monocytes.

Depletion experiments confirmed PDC as crucial IFN- source (FIG. 24D), and furthermore

as potent mediators of CpG-A- and HSV-induced NK cell activation (FIG. 24A) within the

PBMC context. PDC-induced NK cell activation was at least in part due to IFN- production

(FIG. 10, FIG. 12A, FIG. 24D). However, NK cell effector functions did not depend on PDC

(FIG. 24B, C), which stands in contrast to an early study, that indicated a supporting role for

the so called “IFN-producing cells (IPC)” in NK cell-mediated lysis of HSV-1-infected

fibroblasts (Feldman et al., 1992). In addition, our studies showed the importance of

monocytes in NK cell activation (FIG. 24A) and also in IFN- secretion (FIG. 24D).

Monocytes may account for high IFN- levels in different ways. They could contribute

directly by secretion of IFN- itself, as observed for infected monocytes by us (FIG. 19) and

also by others (Linnavuori and Hovi, 1983). However, isolated monocytes only reacted with

IFN- production to infectious, not to UV-inactivated HSV-1, while depletion of monocytes

from PBMC diminished IFN- levels upon stimulation with both HSVINF and HSVUV.

Another possibility would be an indirect contribution of monocytes via secretion of IL-1 and

TNF-, thereby further stimulating CpG-A- and HSV-1-induced IFN- secretion by PDC.

Cytokine neutralization experiments (FIG. 12D) suggest this way of monocyte involvement in

Discussion

79

IFN- production. Furthermore, early studies suggested the dependence of an IFN- response

to HSV-1 on close contact and interactions of IFN-producing cells with other cells within the

cell culture (Rönnblom et al., 1988) and the potential of PBMC-derived cytokines to enhance

HSV-1-induced IFN- secretion by IFN-producing cells (Cederblad and Alm, 1990). Also,

Megjugorac et al. investigated interactions between PDC and HSV-infected monocyte-

derived (mo) DC and could demonstrate induction of IFN- secretion from PDC by HSV-

infected moDC (Megjugorac et al., 2007). Most importantly, we identified monocytes as

indispensable cell population in the induction of NK cell effector functions by HSVINF within

the PBMC context (FIG. 24B, C). Our findings within PBMC appear similar to a study of

PDC-induced NK cell activation, in which NK cell CD69 up-regulation and IFN- production

were induced by soluble factors, whereas degranulation and cytotoxicity were only observed

after direct contact with CpG-stimulated PDC (Benlahrech et al., 2009). In contrast to

Benlahrech et al. we did not find IFN- as major soluble factor for NK cell IFN- production,

but could determine TNF- secretion as important mechanism in the induction of IFN-

(FIG. 12B), which is probably due to the fact that our analyses were conducted with whole

PBMC, not purified PDC and NK cells. The exact process, in which degranulation was

induced, remained elusive.

It is very possible that infected monocytes are directly recognized by NK cells as target cells.

NK cell activation and induction of effector functions could be mediated through various

possible mechanisms. Induction of NK cell cytotoxicity via down-regulation of HLA-C

molecules on productively infected cells was demonstrated for both HSV-1 and HSV-2

(Elboim et al., 2013; Huard and Früh, 2000). Yet, in our studies we observed an overall up-

regulation of HLA-ABC and HLA-E on monocytes inoculated with HSV-1 (FIG. 17,

FIG. 18). MHC-I up-regulation was due to monocytic IFN- secretion (FIG. 19), as

demonstrated by neutralization experiments (FIG. 20, FIG. 21B, C). Unfortunately, we were

not able to investigate HLA-A, HLA-B and HLA-C separately, because no specific antibodies

are available. But the fact that only a minority of HSVGFP-infected monocytes showed

decreased MHC-I expression (FIG. 18) makes recognition of infected monocytes via MHC-I

down-regulation very unlikely. MICA has been shown to be up-regulated on TLR-stimulated

monocytes (Kloss et al., 2008), but MICA/MICB was not induced by HSV-1 in our

Discussion

80

experiments (FIG. 23). Thus, we could exclude direct NK cell activation by infected

monocytes via expression of these stress molecules.

Fitzgerald-Bocarsly et al. demonstrated the expression of immediate early genes as sufficient

to induce NK cell-mediated lysis of HSV-1-infected fibroblasts (Fitzgerald-Bocarsly et al.,

1991), and Chisholm et al. identified ICP0 as effective to trigger lysis of HSV-1-infected cells

by NK cells via the natural cytotoxicity receptors (NCR) NKp30, NKp44, and NKp46

(Chisholm et al., 2007). However, the molecules induced by ICP0 and serving as ligands to

the NCR were not identified in this study. A possible candidate might be B7H6, a molecule

expressed on tumor cells that triggers NK cell cytotoxicity and cytokine secretion via

interaction with NKp30 (Brandt et al., 2009), which was shown to be induced on human

monocytes upon stimulation with TLR ligands and pro-inflammatory cytokines such as IL-1

and TNF- (Matta et al., 2013). Other molecules involved in NK cell-monocyte/macrophage

interaction could be macrophage-expressed CD48 and NK cell-expressed 2B4, which are

involved in NK cell activation after LPS stimulation (Nedvetzki et al., 2007), or macrophage-

expressed activation-induced C-type lectin (AICL) and NK cell-expressed NKp80, which are

involved in NK cell activation after TLR ligand stimulation (Welte et al., 2006), or the

glucocorticoid-induced tumor necrosis factor receptor-ligand (GITRL), which was described

to be involved in the induction of NK cell cytotoxicity by CpG-stimulated PDC (Hanabuchi et

al., 2006) and was shown to be induced on monocytes by staphylococcal enterotoxin B

(Cardona et al., 2006).

While PDC did not mediate NK cell effector functions within the PBMC context (FIG. 24B,

C), they influenced NK cell activation (FIG. 24A). Outside the PBMC context they were

indispensable for NK cell activation, since HSV-1 did not activate purified NK cells directly

(FIG. 25A) (FIG. 34). This observation stands in contrast to a study by Kim et al., where HSV

glycoprotein (g)D peptides directly activated NK cells (Kim et al., 2012). In our co-culture

experiments with HSV-exposed HFF and PDC, neither cell type induced significant CD69 up-

regulation on NK cells by its own. Only in combination, HSV-exposed HFF and PDC

succeeded to strongly activate NK cells (FIG. 25A), which was either caused by synergistic

effects of both cell types on NK cells or mediated by IFN-, which was produced by PDC in

high amounts only in the presence of HFF (FIG. 25B). Dependence of high IFN- production

Discussion

81

by PDC on co-culture with HFF might, similar to IFN- production within PBMC

(FIG. 24D), be due to the need of PDC for close contact to and possible interactions with

other cell populations (Rönnblom et al., 1988). Interestingly, HSV-1-infected HFF somehow

caused down-regulation of CD56 on NK cells (FIG 34), with the effect being enhanced by

PDC (FIG. 26). The significance of this finding remains unclear.

FIG. 34. NK cell activation by HSV-1-infected HFF depends on PDC. HSV-1 does not activate NK cells in a

direct manner, but demands the presence of cells that are stimulated (PDC) or infected (HFF) by HSV-1. HSV-1-

infected HFF induce significant CD69 up-regulation on NK cells only in the presence of PDC, possibly in a type

I IFN-dependent manner, suggesting PDC as important accessory cells for NK cell activation within HSV-1-

infected tissue. Interestingly, NK cells stimulated with HSV-1-infected HFF down-regulate CD56, particularly in

the presence of PDC.

Besides influencing immune responses to HSV-1, PDC might also play a crucial role in

HSV-1 infection by inhibiting viral replication in HSV-1-susceptible cells via secretion of

antiviral cytokines, thereby limiting spread of virus progeny and protecting tissue from

immense damage. We observed an inhibitory effect of PDC-SN in infection experiments with

HFF (FIG. 35). Addition of PDC-SN to HSVGFP-infected HFF clearly decreased HSV-1

replication, evident from reduced green fluorescence of HFF (FIG. 27, FIG. 28B, C).

Discussion

82

Inhibition of HSV-1 replication was most likely mediated by type I IFN, as observed in

monocytes (FIG. 21A), and also demonstrated by others (Härle et al., 2001; Noisakran et al.,

2000).

FIG. 35. PDC supernatants inhibit HSV-1 replication in HFF. Viral infection of and replication in HFF are

diminished in the presence of supernatants derived from HSV-1-stimulated PDC. The inhibitory effect of PDC

supernatants is possibly mediated by type I IFN. These findings evidence the role of PDC as suppressors of

spread of HSV-1 infection within tissue by suppressing viral replication in HSV-1-susceptible cells via secretion

of antiviral cytokines.

Studies of stimulation of PDC and NK cells from HIV-1-infected individuals propose a role

for defective PDC-NK cell interactions in HIV-1-induced immune suppression (Conry et al.,

2009; Reitano et al., 2009), allowing opportunistic or IRIS-related infections. In our case

study, an HIV-1-infected patient suffering from vaginal hyperproliferative lesions due to

HSV-2 and HPV-54 infections exhibited severe functional deficits of PDC as well as NK cells

within the PBMC context. NK cells were only minimally activated by HSV-1 (FIG. 29B)

which appeared to be due to impaired IFN- secretion by PDC (FIG. 29C). Impaired IFN-

secretion upon stimulation with HSV-1 and TLR-7 and TLR-9 agonists might have been

caused by increased CD40-CD40L interactions, as demonstrated by Donhauser et al.

(Donhauser et al., 2012), since sCD40L levels were elevated in the patient (FIG. 29D). Our

findings suggest a role for impaired PDC-NK cell interactions in the severe and treatment-

refractory course of disease in the patient, emphasizing the importance of PDC-NK cell

crosstalk for efficient control of herpesviral infections.

Discussion

83

Altogether, our data propose a model in which the induction of high IFN- levels by HSV-1

within PBMC demands a tight crosstalk between PDC and monocytes involving a positive

feedback loop influenced by type I IFN, TNF- and IL-1 (FIG. 32). Secretion of IL-1 does

not directly influence NK cells, whereas type I IFN and TNF- secreted by both PDC and

monocytes mediate NK cell activation (FIG. 36). IFN- secretion and NK cell activation

alone do not depend on HSV-1 infectivity, whereas only HSVINF, not HSVUV, further induces

NK cell INF- secretion as well as degranulation (FIG. 30). Monocytes, in contrast to PDC,

play a key role in the induction of both NK cell effector functions. HSV-1-induced IFN-

secretion by NK cells is independent of type I IFN, but involves TNF- (FIG. 36), which is

only produced in sufficient amounts in response to HSVINF, not HSVUV. In contrast, NK cell

degranulation is independent of all three cytokines tested and either involves other cytokines

produced by monocytes or is mediated by direct cell:cell interactions between NK cells and

monocytes (FIG. 36). Presumably, NK cells recognize HSV-1-infected monocytes as target

cells via mechanisms other than monocytic MHC-I down-regulation or MICA/MICB

expression. While monocytes are particularly important in the activation of effector NK cells,

PDC appear to contribute to immune control early during infection by protecting HSV-1-

susceptible tissue as they suppress viral replication via secreted antiviral cytokines, probably

type I IFN, and therefore limit viral spread (FIG. 36).

Our data may stimulate further studies investigating cell surface molecules as well as

cytokines involved in the crosstalk between PDC, monocytes and NK cells. Deciphering the

mechanisms that induce functional effector NK cells is important as all three cell types are

among the first cells to infiltrate herpetic lesions and thereby may contribute to the efficient

control of primary and recurrent herpes simplex virus infections.

Discussion

84

FIG. 36. Monocytes mediate HSV-1-induced activation of effector NK cells, while PDC limit HSV-1

replication within infected tissue. While depletion of PDC as well as monocytes greatly diminishes secretion of

IFN-2a/2b (IFN-) and CD69 up-regulation on NK cells, only depletion of monocytes prevents NK cell

effector functions IFN- secretion and degranulation, identifying monocytes as crucial accessory cell population

for HSV-1-induced activation of effector NK cells within PBMC. Both type I IFN and TNF- are involved in

CD69 up-regulation, whereas only TNF- impacts IFN- secretion. Degranulation is independent of type I IFN,

TNF- and IL-1 and is possibly mediated by infected monocytes via direct cell contact. The ability of PDC

supernatants to inhibit HSV-1 replication in HFF proves PDC as important cell population in the limitation of

spread of infection within tissue via secretion of antiviral cytokines.

Abbreviations

85

7 Abbreviations

Abbreviation Full length spelling

AC accessory cell(s)

ADCC antibody-dependent cellular cytotoxicity

AICL activation-induced C-type lectin

APC allophycocyanin

ARN acute retinal necrosis

B bone marrow-derived

BDCA blood dendritic cell antigen

BSA bovine serum albumin

C Celsius

CCL chemokine (C-C motif) ligand

CD cluster of differentiation

cm centimeter

CXCL chemokine (C-X-C motif) ligand

Cy cyanine

D day(s)

DMEM Dulbecco`s Modified Eagle Medium

DNA deoxyribonucleic acid

DNAM DNAX accessory molecule

DPBS phosphate buffered saline without calcium or magnesium

ELISA enzyme-linked immunosorbent assay

EDTA ethylenediaminetetraacetic acid

FACS fluorescence-activated cell sorting

FasL Fas ligand

Fc fragment, cristallizable

FcRIIIA low affinity Fc receptor IIIA

FCS fetal calf serum

FITC fluorescein isothiocyanate

FSC forward scatter

g glycoprotein

GFP green fluorescent protein

GITRL glucocorticoid-induced tumor necrosis factor receptor-ligand

h hour(s)

HIV human immunodeficiency virus

HFF human foreskin fibroblast(s)

HLA human leukocyte antigen

Abbreviations

86


HPV human papilloma virus

HRP horseradish peroxidase

HSV herpes simplex virus

ICP infected cell polypeptide

IFN interferon(s)

Ig immunoglobulin

IL interleukin

IPC interferon producing cell(s)

IRIS immune reconstitution inflammatory syndrome

J Joule

KIR killer cell immunoglobulin-like receptor

L ligand

l liter

MACS magnetic-activated cell sorting

MDA5 melanoma differentiation-associated protein 5

MFI median fluorescence intensity

MHC major histocompatibility complex

MIC MHC class I polypeptide-related sequence

min minute(s)

µl microliter

ml milliliter

µm micrometer

mo monocyte-derived

MOI multiplicity of infection

n nano

NCAM neural cell adhesion molecule

NCR natural cytotoxicity receptor(s)

NKG2D natural killer group 2, member D

NO nitric oxide

NK cell natural killer cell

OD optical density

ODN oligodeoxynucleotides

PBMC peripheral blood mononuclear cell(s)

PBS phosphate buffered saline

PDC plasmacytoid dendritic cell(s)

PE phycoerythrin

PFA paraformaldehyde

Abbreviations

87


p.i. post infection

p.p. post purification

p.s. post stimulation

R receptor

rh recombinant human

RIG-I retinoic acid-inducible gene I

ROS reactive oxygen species

RPMI 1640 Roswell Park Memorial Institute 1640 Medium

s soluble

SSC sideward scatter

T thymus-derived

TCID tissue culture infective dose

Th T helper

TLR toll-like receptor

TNF tumor necrosis factor

TRAIL TNF-related apoptosis-inducing ligand

Tris tris(hydroxymethyl)aminomethane

ULBP UL-16-binding protein

UV ultraviolet light

VSB virus standard buffer

VZV varicella zoster virus

W Watt

References

88

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Publications

99

9 Publications

Vogel K, Thomann S, Vogel B, Schuster P, Schmidt B. Both plasmacytoid dendritic cells and

monocytes stimulate natural killer cells early during human HSV-1 infections. Immunology. 2014 Dec;

143(4):588-600.

Vogel B, Tennert K, Full F, Ensser A. Efficient generation of human natural killer cell lines by viral

transformation. Leukemia. 2014 Jan; 28(1):192-5.

Tennert K, Schneider L, Bischof G, Korn K, Harrer E, Harrer T, Schmidt B; German Competence

Network HIVAIDS. Elevated CD40 ligand silences α interferon production in an HIV-related immune

reconstitution inflammatory syndrome. AIDS. 2013 Jan 14; 27(2):297-9.

Schuster P, Boscheinen JB, Tennert K, Schmidt B. The Role of Plasmacytoid Dendritic Cells in

Innate and Adaptive Immune Responses against Alpha Herpes Virus Infections. Adv Virol. 2011;

2011:679271.

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Role of plasmacytoid dendritic cells and other accessory ... · Role of plasmacytoid dendritic cells and other accessory cells in the activation of human natural killer cells by herpes