Role of Complement Receptors in the Induction of Retroviral-Specific CTL Responses by Dendritic Cells” Supervisor: Prof

Role of complement receptors in the induction of retroviral- specific CTL responses by dendritic cells

Doctoral thesis submitted to the Medical University of Innsbruck

for obtaining the academic degree

Doctor of Philosophy (PhD)

Muneeb Ahmad Idrees

Supervisor:

Univ.-Prof. Dr. Heribert Stoiber

Department of Hygiene, Microbiology and Social Medicine

Division of Virology

Medical University of Innsbruck

Innsbruck, March 2017

DEDICATION

This thesis is dedicated to my wonderful parents, who have raised me to be the person I am today. You have been with me every step of the way, through good times and bad. Thank you for all the unconditional love, guidance, and support that you have always given me, helping me to succeed and instilling in me the confidence that I am capable of doing anything I put my mind to.

Thank you for everything. I love you!

Table of Contents

SUMMARY ...... 1

1. INTRODUCTION ...... 2

1.1. Complement System ...... 2

1.1.1. Complement activation pathways ...... 2

1.1.1.1. Classical pathway ...... 3

1.1.1.2. Lectin pathway ...... 4

1.1.1.3. Alternative pathway ...... 5

1.1.2. Effector functions mediated by complement system ...... 5

1.1.3. Protection of host by regulation of complement system ...... 7

1.1.4. Complement evasion by pathogens ...... 9

1.1.5. Complement receptors ...... 10

1.1.5.1. Complement receptor type 1 (CR1) ...... 10

1.1.5.2. Complement receptor type 2 (CR2) ...... 12

1.1.5.3. Complement receptor type 3 (CR3) ...... 12

1.1.5.4. Complement receptor type 4 (CR4) ...... 14

1.1.6. Complement in regulation of adaptive immunity ...... 14

1.2. Dendritic cells ...... 14

1.2.1. Maturation of DCs ...... 15

1.2.2. Role of DCs in the induction of T cell responses ...... 16

1.2.2.1. MHC-I restricted antigen presentation (Endogenous pathway) ...... 16

1.2.2.2. MHC-II restricted antigen presentation (Exogenous pathway) ...... 16

1.2.2.3. Cross-presentation ...... 17

1.3. Friend Virus ...... 18

1.3.1. Friend Virus as a platform to investigate immune response in retroviral infections ...... 19

1.3.2. Friend virus induced erythroleukemia ...... 19 I

1.3.3. Advantages of FV as retroviral infection model ...... 20

1.3.4. Comparison of FV and HIV-1 ...... 21

1.3.5. Morphology of FV ...... 22

1.3.5.1. Replication cycle of FV ...... 23

1.3.5.2. Maturation of FV virion ...... 24

1.3.5.3. Genome of FV ...... 25

1.3.5.4. Proteins of FV ...... 26

1.3.5.4.1. Gag ...... 26

1.3.5.4.2. Pol ...... 27

1.3.5.4.3. Env ...... 27

1.3.6. Pathology of Friend disease: EpoR and gp55 Molecular interactions ...... 29

1.3.7. Resistance to FV infection: role of SF-Stk ...... 30

1.3.8. Immune responses in FV infection ...... 32

1.3.8.1. Role of CD8+ T cells in FV-specific immunity ...... 32

1.3.8.2. Role of CD4+ Regulatory T cells in FV-specific immunity ...... 32

1.3.8.3. Role of CD4+ Helper T cells in FV-specific immunity ...... 33

1.3.8.4. Role of antibodies and B cells in FV-specific immunity ...... 34

1.3.8.5. Role of Natural Killer T (NKT) cells in FV-specific immunity...... 34

2. AIMS OF THE STUDY ...... 36

3. MATERIALS AND METHODS ...... 37

3.1. Materials ...... 37

3.1.1. Equipment ...... 37

3.1.2. Chemicals and media ...... 38

3.1.3. Antibodies ...... 38

3.1.4. Buffers and solutions ...... 39

3.1.5. Animals ...... 40

3.2. Methods ...... 40

3.2.1. Mice ...... 40

3.2.2. Cultivation of Mus dunni cells ...... 40

3.2.2.1. Storage of Mus dunni cells ...... 40

3.2.3. Virus stocks production ...... 41

3.2.4. Quantification of virus by infectious centers (IC) assay ...... 41

3.2.5. Flow cytometry ...... 42

3.2.6. Generation of mouse bone marrow-derived DCs (bmDCs)...... 42

3.2.7. Opsonization of FMuLV ...... 43

3.2.8. Real-time PCR ...... 43

3.2.9. Virus capture assay ...... 44

3.2.10. In vitro productive infection of bmDCs for IC assay...... 44

3.2.11. In vitro infection of bmDCs for FACS analysis ...... 44

3.2.12. Isolation of CD8+ T cells ...... 45

3.2.13. Co-culture of FMuLV loaded bmDCs with FV-specific CD8+ TCRtg T cells .... 45

3.2.14. Statistical analysis ...... 45

4. RESULTS ...... 46

4.1. FMuLV propagation and IC assay ...... 46

4.2. Loading dunni cells with FMuLV- Viability and Infection level ...... 47

4.3. Generation of bmDCs and their phenotypical characterization ...... 49

4.4. Flow cytometry analysis of FMuLV-loaded bmDCs ...... 51

4.5. Viability and infection level of FMuLV loaded bmDCs- FACS analysis ...... 52

4.6. Productive infection of bmDCs ...... 54

4.7. RT-PCR of FMuLV-loaded bmDCs SNs ...... 55

4.8. Opsonization of FMuLV-Virus capture assay ...... 56

4.9. Productive infection of bmDCs with differntially opsonized FMuLV ...... 57

4.10. Characterization of CD11b-/- and CD11c-/- mice derived DCs ...... 59

4.11. Maturation behavior of CD11b-/- and CD11c-/- mice derived DCs ...... 60

III

4.12. CD8+ T cell activation by CD11b-/- and CD11c-/- mice derived DCs ...... 61

5. DISCUSSION ...... 63

6. REFERENCES ...... 67

CURRICULUM VITAE ...... 85

ACKNOWLWDGEMENTS ...... 88

Summary

SUMMARY

In the present study, we investigated the role of complement receptors in the induction of cytotoxic T lymphocyte (CTL) responses mediated by dendritic cells (DCs) upon infection with retroviruses. For this purpose, Friend virus (FV) was adopted as a mouse retrovirus infection model.

DCs are the most potent antigen presenting cells (APCs) and therefore, are able to efficiently induce CTL responses. DCs are known to express complement receptors (CRs) on their surface, which enable them to interact with complement opsonized viruses. Such interactions might affect the infection behavior of DCs and their antigen presenting capacity which consequently may influence the induction of CTL responses. Therefore, as our first aim we studied the relation between opsonization of retrovirus and the infection behavior of DCs at different viral loads. We showed that DC infection with higher viral doses resulted in the loss of infectivity of the output virus. Further, the complement-opsonized virus lead to improved infection of DCs, while IgG-opsonization of virus, in contrast, resulted in a decrease in the infection of DCs. Our findings suggest that virus activates DCs in a complement-dependent manner in order to mount cellular immune responses.

The second goal of our study was to determine the specific roles of CR type 3 (CR3) and CR type 4 (CR4) in the induction of CTL responses upon retrovirus infection. The two complement receptors are co-expressed on DCs and their ligand specificity resembles greatly. To define their specific roles, we used CD11b (CR3) and CD11c (CR4) knock-out mice- derived DCs. It was observed that both complement opsonized or non-opsonized virus loaded- DCs derived from CD11c-/- mice were able to activate CD8+ T cells in a fashion comparable to those derived from wild type (wt) mice. However, virus-loaded DCs derived from CD11b-/- mice were able to activate a significantly lower number of CD8+ T cells compared to those derived from wt mice. Our findings indicated that CR3 plays a crucial role in the infection of DCs and subsequently in the induction of cellular immune responses. Our results may provide valuable information about the approaches to target antigens to DCs in order to enhance vaccine efficacy or to develop novel and improved vaccine candidates.

Introduction

1. INTRODUCTION

1.1. Complement System The complement system is a very old defense tool which developed approximately 600-700 million years ago, long before the emergence of Immunoglobulins (Kolev et al., 2014; Sunyer et al., 1998). Complement system was traditionally considered a part of innate immunity which “complements” the antibodies in killing of invading pathogens (Mathern and Heeger, 2015). Today, we know that complement orchestrates immunological processes ranging far beyond merely elimination of danger (Ricklin et al., 2010). Complement is one of the first lines of defense in innate immunity and underlies one of the main effector functions of antibody-mediated immunity (Walport, 2001a; Zipfel and Skerka, 2009). Studies over the years have shown that complement play its role in almost each step of immune response. It is a complex innate surveillance system, playing pivotal role in homeostasis, inflammation and host defense (Merle et al., 2015b). Complement system has three major physiological roles: host defense against infections, bridge between innate and adaptive immunity (Carroll, 2004), and disposal of immune complexes and apoptotic cells (Walport, 2001a).

Complement comprises of more than 50 soluble proteins and surface-bound receptors and regulators that interact with various cells and mediators of the immune system (Freeley et al., 2016; Ricklin et al., 2010). Complement proteins constitute around 15% of plasma globulin fraction, amounting more than 3 g per liter plasma (Walport, 2001a). This group of proteins is organized into a hierarchy of proteolytic downstream processes which begins with the recognition of pathogens and results in production of efficient pro-inflammatory mediators (anaphylatoxins, C3a and C5a) which attract and activate leukocytes; opsonization in which various opsonins (C3b, iC3b and C3d) ‘coat’ the pathogenic surface through covalent bonding in order to facilitate removal of target cells; and targeted lysis of pathogens or damaged cells (opsonized) through the formation of membrane attack complex (MAC, C5b-9) (Dunkelberger and Song, 2010; Noris and Remuzzi, 2013).

In the nomenclature, the smaller cleavage products are named as ‘a’, e.g., C5a, and the larger cleavage fragments are designated as ‘b’, e.g., C5b (Mathern and Heeger, 2015).

1.1.1. Complement activation pathways The major rule for complement is that everything that has no specific protection must be attacked. Host cells bear “don’t attack me” molecules which are either recruited by plasma to the cell membrane or thy expressed by cell. Any cell, microbe or foreign material lacking 2

Introduction these molecules represents an “activating surface” and triggers the complement activation (Merle et al., 2015b). Complement can be activated through three different pathways: classical, lectin and alternative. All of these three pathways share the common step of activating the component C3, however, they differ in the nature of recognition (Carroll, 2004). All these three pathways after converging at the cleavage of C3 then trigger the generation of the MAC, resulting in cell lysis (Fig. 1.1). The initiators of all the pathways are summarized in Table 1.1.

Table 1.1: Activators of complement activation pathways (Walport, 2001a).

Activation Pathway Activators

Classical Apoptotic cells, immune complexes, certain viruses and gram- negative bacteria

Lectin Microorganisms with terminal mannose groups

Alternative Several bacteria, fungi, tumor cells, viruses

1.1.1.1. Classical pathway The classical pathway is initiated by antigen-antibody immune complexes being recognized and bound by C1q. This is a way in which C1q acts as a bridge between innate and adaptive immunity (Walport, 2001b). This interaction results in the sequential activation of C1r and C1s, followed by the cleavage of C4 and C2 by C1s into inactive peptides (C4a and C2a) and active peptides (C4b and C2b) which assemble to form C3 convertase (C4bC2b). In some older texts, C2a is referred to as the larger fragment of C2, however, the current nomenclature which has been widely accepted, the larger fragment is denoted as C2b, and the same is used in this manuscript. C3 convertase cleaves C3, the central component of the complement cascade, into C3a (anaphylatoxin) and C3b. Binding of C3b to C4b generates C5 convertase (C4b2b3b). The assembly of C5b-9 begins when C5 is cleaved into C5a and C5b and C5b6 complex is formed that can bind reversibly to the cell membrane via ionic as well as hydrophobic linkages. The subsequent interaction of C7, C8, and C9 with C5b6 results in the formation of a superamolecular C5b-9 complex, referred to as MAC. This complex is able to induce cell lysis (Markiewski and Lambris, 2007; Rus et al., 2005; Tegla et al., 2011).

Introduction

1.1.1.2. Lectin pathway The lectin pathway resembles the classical pathway in a sense that the activation of this pathway also leads to the assembly of C4bC2b, C3 convertase. But, opposed to relying on antigen-antibody complexes recognition, this pathway employs plasma proteins, such as mannose-binding lectin (MBL) and ficolins, to recognize carbohydrate groups found on the surface of a diverse range of microbes (Noris and Remuzzi, 2013; Tegla et al., 2011). MBL has a similar general structure to C1q, however it has multiple oligomeric forms, i.e., trimers, tetramers, etc. (Merle et al., 2015a). Attachment of MBL or ficolin to carbohydrate molecules on pathogens surface results in the activation of MBL-associated serine proteases (MASP-1, MASP-2, MASP-3), thus initiating their esterase activity. MASPs cleave and activate C4 and C2 generating C4bC2b, C3 convertase, in a reaction analogous to the classical pathway (Dunkelberger and Song, 2010; Rus et al., 2005).

Fig 1.1: Activation of complement system. Adapted from (Mathern and Heeger, 2015).

Introduction

1.1.1.3. Alternative pathway Even though the classical and lectin pathways are activated upon recognizing the pathogens, the alternative pathway is normally active at low levels in the host. This is referred to as ‘tick- over theory’ which makes the system able to stay ready for quick and robust activation (Bexborn et al., 2008; Merle et al., 2015a). The alternative pathway is activated when factor B bound to the C3b derived from classical pathway or to C3(H2O), the spontaneously hydrolyzed from of C3; is cleaved by a serine protease, factor D, into Ba and Bb. This generates C3bBb or C3(H2O)Bb, known as alternative pathway C3 convertase, which similar to the classical C3 convertase, can cleave C3 into C3a and C3b. Further steps lead to the formation of MAC and microbe death in a way analogous to classical and lectin pathways (Noris and Remuzzi, 2013; Tegla et al., 2011).

1.1.2. Effector functions mediated by complement system Regardless of the pathway by which complement is activated, it converges on the initiation of three effector pathways that make it possible for the complement to defend the host: (a) direct lysis of target cell by MAC assembly; (b) opsonization of the target and its clearance by means of complement receptors on phagocytic cells; and (c) signaling as well as priming the immune system by producing pro-inflammatory anaphylatoxins (Fig. 1.2).

During the complement activation the cleavage of C5 by C5 convertase leads to the generation of C5b which makes the basis for assembly of MAC. Firstly, C5b links itself to the target surface and C6 and C7. C8 binds to this complex and is also partially inserted into the target membrane. This leads to the association of C9 to the complex and also insertion of C9 molecule into the lipid bilayer. Later 12-15 molecules of C9 form a stably inserted pore of approximately 10nm diameter, in the membrane. The pore formation results in the lysis of the surface and killing of the pathogen (Dunkelberger and Song, 2010; Mathern and Heeger, 2015).

Complement opsonized antigens are preferentially recognized by complement receptors (CRs) expressed on macrophages and other antigen presenting cells (APCs), such as dendritic cells (DCs) and B cells. The opsonization process initiates with the generation of C3b fragments in the three activation pathways. C3b fragments and its cleavage products ‘tag’ the foreign bodies which are then recognized by CRs 1-4. Fc region of antibody binds to the Fc receptors. Interaction of complement receptors to the opsonized particles triggers their uptake by phagocytes, for instance, neutrophils and macrophages (Dunkelberger and Song, 2010; Mathern and Heeger, 2015). 5

Introduction

C3a and C5a, the cleavage products of C3 and C5, respectively, are anaphylatoxins which are potent pro-inflammatory molecules. Interaction of anaphylatoxins to their cognate receptors, C3aR and C5aR, induces vasodilation and cytokine and chemokine release. They are also involved in chemoattraction of macrophages and neutrophils, activation of macrophages to stimulate killing of engulfed pathogens, and activation, expansion and survival of T cells and APCs (Guo and Ward, 2005; Klos et al., 2009; Kwan et al., 2013; van der Touw et al., 2013).

Fig 1.2: Effector functions of complement system. Adapted from (Dunkelberger and Song, 2010).

Introduction

1.1.3. Protection of host by regulation of complement system The powerful effector functions of complement have the potential to severely harm the host; therefore, it is critical to tightly regulate the complement activation. The activated C3b and C4b can bind to pathogens as well as to host cells where they may cause tissue damage and inflammation (Freeley et al., 2016). For this reason several steps in activation of complement are checked by inhibitors so as to make the final system a sophisticated homeostatic balance between efficient recognition and damage of pathogens and the minimization of tissue damage. There is a variety of both plasma and membrane-bound inhibitory proteins which work to regulate the location and activity of complement (Fig. 1.3). Several of the regulator proteins have additional roles other than complement. Primarily, regulation of complement occurs at two steps during the activation cascade, either in the assembly and enzymatic activity of convertases, or during the MAC assembly (Liszewski et al., 1996). The mechanisms of complement regulation are summarized in Fig. 1.4.

Fig 1.3: Regulators of complement system. Adapted from (Zipfel and Skerka, 2009).

Complement factor I, a serine protease, is one of the key plasma regulators. Factor I prevents C3b and C4b from forming active convertases by catabolizing into inactive fragments, for instance, iC3b, C3c and C3dg (Nilsson et al., 2011; Sim et al., 1993). Factor I needs cofactors for its proteolytic activity to avoid non-specific C3b degradation. The cofactors are complement receptor 1 (CR 1 or CD 35), membrane cofactor protein (MCP or CD46), and Factor H (Sharma and Pangburn, 1996; Turnberg and Botto, 2003). 7

Introduction

Factor H is another plasma regulator that is a single chain glycoprotein composed of 20 short consensus repeat units, circulating at a concentration of about 400 mg/L (Pickering and Cook, 2008; Zipfel and Skerka, 1994). Host-specific protection by Factor H is achieved by its binding to sialic acid and heparin which is a surface component of eukaryotic cells but not of prokaryotes (Blackmore et al., 1998; Turnberg and Botto, 2003).

An example of complement inhibitors which possess decay-accelerating activity for convertases is decay-accelerating factor (DAF, CD55), which acts by shortening the half-life of pre-formed convertases. Other complement inhibitors from this category include CR 1 that acts as a cofactor for factor I mediated inactivation of C3b (Khera and Das, 2009), factor H, C4-binding protein (C4BP) and rodent-specific regulator (Turnberg and Botto, 2003).

The final level of complement regulation is to prevent the assembly of MAC by means of membrane-bound CD59 (Protectin) or fluid-phase Vitronectin, Clusterin, complement factor H–related protein 1 (CFHR1), S protein inhibitors (Heinen et al., 2009; Meri et al., 1990; Preissner and Seiffert, 1998; Schwarz et al., 2008; Turnberg and Botto, 2003).

Fig 1.4: Mechanisms of complement regulation. Modified from (Dunkelberger and Song, 2010; Mathern and Heeger, 2015).

Introduction

Anaphylatoxins, C3a and C5a, are regulated by serum carboxypeptidases, such as carboxypeptidase N, B and R. The formation of ‘des-Arg’ forms of the anaphylatoxins due to the removal of their N-terminal arginine residue diminishes their ability to bind to the cognate receptors, C3aR and C5aR (Dunkelberger and Song, 2010; Mueller-Ortiz et al., 2009).

1.1.4. Complement evasion by pathogens The crucial determining factor in the virulence of pathogens is their ability to evade the host immune system. These escape strategies are mostly focused on the complement machinery which is the first line of defense against pathogens. In this regard, pathogens interfere with the complement at all levels including regulation, opsonization, phagocytosis and assembly of MAC (Rooijakkers and van Strijp, 2007). As shown in Fig. 1.5, he escaping strategies can be broadly categorized as: recruitment or imitating of complement regulators, inhibition of complement proteins, enzymatic degradation for inactivation of complement components (Lambris et al., 2008). Additionally, several pathogens have also evolved passive escape features, for instance, Gram-positive bacterial cell wall cannot be lysed by MAC (Joiner et al., 1983).

Most pathogens express surface proteins which interact with the complement regulators of host which as a result block the protective host immune response against the microbe. Viruses of the families Poxviridae and Herpesviridae, encode for regulators for complement activation (RCAs) in their genome, while those belonging to Togaviridae and Retroviridae are able to incorporate host complement regulators in the viral envelope thus up-regulating the expression of these regulator proteins in the infected cell, ultimately escaping the immune response (Favoreel et al., 2003; Stoiber et al., 2008a; Stoiber et al., 2003). Complement regulators recruitment has also been shown in bacteria (e.g., Escherichia coli, Borrelia burgdorferi, Staphylococcus aureus) (Lambris et al., 2008; Rooijakkers et al., 2005), fungi (Candida albicans) (Meri et al., 2004; Meri et al., 2002) and parasites (Echinococcus spp.) (Inal, 2004). An example of direct inhibition of complement is Staphylococcal superantigen-like protein-7 (SSL-7) produced by S. aureus that binds to C5 resulting in complement-mediated lysis (Langley et al., 2005). CD59-like protein from B. burgdorferi binds to C9 inhibiting the formation of MAC (Frank, 2001). Herpes simplex virus 1 and 2 have transmembrane glycoproteins gC1 and gC2, respectively, and accelerate the decay of lectin pathway C3 convertase (Lubinski et al., 1998).

Introduction

The cleavage of complement proteins into inactive fragments is also used by pathogens, e.g., Pseudomonas elastase degrades C1q in classical pathway (Rooijakkers and van Strijp, 2007); proteases from Serratia mercescens and streptococci cleave C5a (Chmouryguina et al., 1996; Oda et al., 1990). The complement evasion strategies delay or block the complement immune responses which otherwise would remove the pathogenic organism. Nevertheless, a persisting pathogen activates other immune reactions (Zipfel and Skerka, 2009).

Fig 1.5: Evasion of pathogens from complement actions. Adapted from (Lambris et al., 2008).

1.1.5. Complement receptors Complement receptors are proteins expressed on the membrane surface of cells of immune system. These receptors specifically interact with other complement factors resulting in the elimination of antigens. Some of the well-known complement receptors are summarized in Table 1.2 and Fig. 1.6. Complement receptors for C3b and its derivatives include complement receptor (CR) type 1, CR type 2, CR type 3 and CR type 4.

1.1.5.1. Complement receptor type 1 (CR1) CR1 or CD35 is a 210-290 kDa membrane glycoprotein composed of 30 short consensus repeats (SCR) which is specific for C3b, C4b and, with lower affinity, iC3b (Leslie, 2001). The first 28 SCRs are arranged in four long homologous repeats (LHRs) (Wong et al., 1989). LHR-A interacts with C4b, while LHR-B and LHR-C interact with both C3b and C4b. LHR- D binds to MBL (Khera and Das, 2009) (Fig. 1.7). 10

Introduction

Table 1.2: Complement receptors. Modified from (Leslie, 2001; Mathern and Heeger, 2015; Zipfel and Skerka, 2009).

Complement Synonyms Ligand Function Cell Type Receptor C5aR CD88 C5a Leukocyte Monocytes, chemoattraction, dendritic cells, degranulation neutrophils, T cells C3aR None C3a Immune cell recruitment Neutrophils, and inflammation monocytes, eosinophils, T cells CR1 CD35, Immune C3b, iC3b, Immune complex Leukocytes, adherence C4b, C1q clearance, augmentation of monocytes, receptor phagocytosis, regulation of RBCs, follicular C3 degradation dendritic cells CR2 CD21, Epstein- iC3b, C3d, Proliferation of B cells, B cells, T cells, Barr virus C3dg activation of alternative follicular receptor pathway dendritic cells CR3 CD11b/CD18, iC3b, iC3b enhances contact of Monocytes, MAC1, αMβ2 factor H opsonized particles leading macrophages, integrin to phagocytosis neutrophils, NK cells, dendritic cells, T cells CR4 CD11c/CD18, iC3b iC3b-mediated Monocytes, p150/95, αXβ2 phagocytosis, leukocyte macrophages, integrin migration dendritic cells

Fig 1.6: Complement surface receptors. Adapted from (Zipfel and Skerka, 2009).

Introduction

1.1.5.2. Complement receptor type 2 (CR2) CR2 or CD21 is a 145 kDa membrane glycoprotein composed of 16 SCRs in humans and 15 SCRs in mice (Moore et al., 1987) (Fig. 1.7). The major ligand for CR2 is C3d, but it can also interact with iC3b and C3dg (Erdei et al., 2009). CR2 plays a role in antibody maturation and the induction of B cell memory. It has a vital function of bridging the innate and adaptive immune responses.

Fig 1.7: Structure of CR1 and CR2 (Leslie and Hansen, 2009).

1.1.5.3. Complement receptor type 3 (CR3) CR3, Mac-1 or CD11b/CD18 belongs to the integrin family consisting of non-covalently associated α and β chains. As illustrated in Fig. 1.8, CR3 is a β2 integrin that consists of

CD11b as α chain (also termed as αM) and CD18 as β chain (also called β2) (Sanchez-Madrid et al., 1983; Speth et al., 1997). CD11b chain is a transmembrane protein of 165 kDa with seven tandem repeats of 65 amino acids at its amino terminus. A 200 amino acids sequence, known as interactive domain or I-domain, is present between domains II and III. This I- domain carries the binding sites for most of the ligands (Diamond et al., 1993; Ueda et al., 1994). A lectin-binding domain exists on the carboxy-terminal of I-domain with the ability to interact with certain polysaccharides including β-glucan (Thornton et al., 1996; Vetvicka et

Introduction al., 1996). There is a short cytoplasmic region with only 19 amino acids and carries residues as potential targets for phosphorylation (Speth et al., 1997).

CD18, β2 integrin chain is a 95 kDa transmembrane protein with a short cytoplasmic tail comprising of tyrosine, threonine and serine residues responsible for phosphorylation of Cd18 upon the stimulus. The α and β chains are anchored in the membrane independently (Speth et al., 1997).

CR3 has been shown to interact with a large variety of ligands, for instance iC3b, Intercellular Adhesion Molecule-1 and -2 (ICAM-1 and ICAM-2), factors of blood coagulation and molecules of microbial origin, e.g., Mycobacterium tuberculosis, Candida albicans, or Klebsiella pneumonia. Mac-1 is able to interact with LPS, therefore, proving its role in innate immune responses (Ehlers, 2000).

CR3 also plays pivotal role in phagocytosis by interacting to pathogens either by LPS or CR3- stimulatimg polysaccharides or by opsonizing iC3b fragments on the antigen surface. The recognition of iC3b by neutrophils or NK cells via I-domain is essential for triggering phagocytosis and degranulation (Vetvicka et al., 1996). Further, CR3 has been shown to be also involved in cellular aggregation, migration and chemotaxis (Speth et al., 1997).

Fig 1.8: Structure of CR3. Adapted from (Speth et al., 1997).

Introduction

1.1.5.4. Complement receptor type 4 (CR4) Similar to CR3, CR4 (CD11c/CD18) also belongs to the family of β2 integrins. It shares the

CD18 chain with CR3 (Lanier et al., 1985). The α chain CD11c, also known as αX, is a 150 kDa transmembrane protein which is highly homologous to the α chain (CD11b) of CR3. This striking homology is a reflection of their closely related functionality (Speth et al., 1997).

Like CR3, CR4 also binds mainly to iC3b (Myones et al., 1988; Xie et al., 2010). CR4 in comparison to CR3 is less well characterized but its ligands overlap those of CR3. CR4 is expressed by monocytes, macrophages, granulocytes, B cells, dendritic cells and some T cells (Bilsland et al., 1994; Xie et al., 2010). In addition to iC3b, CR4 interacts with iC3b- opsonized particles, fibrinogen, ICAM-1 and ICAM-2 and is also responsible for the adhesion of neutrophils and monocytes to endothelium and other cells (Bilsland et al., 1994; Choi and Nham, 2002; Keizer et al., 1987; Stacker and Springer, 1991; te Velde et al., 1987).

Though CD11c is also expressed on sub-populations of mice NK cells, T cells and B cells, it is preferentially expressed on myeloid DCs and it is thus frequently stated as DC-specific marker (Caminschi et al., 2007; Racine et al., 2008; Singh-Jasuja et al., 2013; Vinay and Kwon, 2010).

1.1.6. Complement in regulation of adaptive immunity Complement system is involved in regulating the B and T cells responses. The proliferation of B cells is influenced by the presence of CR1 and CR2 on B cells (Fingeroth et al., 1989).

A dual role is played by complement receptors in T cell activation; either direct effect of complement receptors on T cells or indirect effect cytokine triggering of T cells by complement receptor-activated B cells (Speth et al., 1997). The indirect activation of T cells by complement receptors is more prominent. Binding of C3b or C4b increases proliferation and cytotoxic effects of T cells by targeting opsonized antigen on CR1 and CR2 of B cells (Arvieux et al., 1988).

1.2. Dendritic cells DCs are potent antigen presenting cells (APCs) which play a crucial role in innate responses as well as in adaptive immune response by priming the immune responses (Steinman and Hemmi, 2006). DCs were first described in 1973 by Ralph Steinman, isolated from spleen of mouse (Steinman et al., 1975; Steinman and Cohn, 1973, 1974; Steinman et al., 1974). DCs have different phenotypes which are distributed all over the body. At the site of pathogen

Introduction entry or injury, DCs differentiate into immature DCs. The diversity of the roles of DCs is dependent on their subsets as well as on their state of activation (Banchereau et al., 2000).

DCs are efficient stimulators of T and B cells. They are exceptionally equipped with the ability to activate naïve T lymphocytes, therefore termed as “professional” APCs. DCs are capturing antigens either by micropinocytosis, phagocytosis, or receptor-facilitated endocytosis (Toll-like receptors (TLR), Fc receptors, complement receptors, and C-type lectin receptors (CLR) like DEC-205, DC-SIGN and mannose receptors) (Hubo et al., 2013; Tacken and Figdor, 2011).

DCs have the ability to synthesize a variety of complement components which have a crucial effect on the activation of DCs, their antigen presentation capacity and ability to stimulate T cells (Peng et al., 2006; Strainic et al., 2008).

1.2.1. Maturation of DCs In most of the tissues, DCs are residing in a so-called “immature” state, which are not able to stimulate T cells. However, these DCs are extremely efficient in capturing antigens, and the antigens in turn induce full maturation and mobilization of DCs to draining lymph nodes. They have low MHC, CD40 and CD54 molecules, while lot of Fc and Fc receptors (Banchereau and Steinman, 1998).

Fig. 1.9: Maturation of DCs. Modified from (Banchereau and Steinman, 1998; Hubo et al., 2013).

Introduction

The maturation of DCs begins with antigen encounter, which is associated with morphological change, upregulation of co-stimulatory molecules, for instance, CD40, CD80 and CD86, and upregulation of MHC-II expression; and down-regulation of endocytic receptors. The phenotype changes dramatically, antigen uptake machinery disappears and antigen presenting and T cell stimulatory devices appear (Banchereau and Steinman, 1998; Cavanagh and Von Andrian, 2002) (Fig. 1.9).

1.2.2. Role of DCs in the induction of T cell responses B cells can directly recognize antigens through their B cell receptors, however, T cells require the antigen to be processed and presented to them by APCs. T-cell antigen receptors (TCR) interact with the antigen fragments presented by the molecules of major histocompatibility complex (MHC) on the surface of APC. The major role played by DCs is the induction of T cell responses. The efficiency of signal received by T cells is 100 folds increased if the costimulatory molecules are present in comparison to antigen recognition by TCR alone (Lanzavecchia and Sallusto, 2001). DCs have the ability to modify both CD4+ and CD8+ T cell responses. DCs are also important for the T cell-dependent antibody responses development. DCs are able to activate CD4+ T cells which in turn interact with B cells for antibody response. The antibody production may also be directly influenced by DCs via CD40 and IL-2 (Dubois et al., 1997).

DCs efficiently present antigens to T cells via MHC class I or II molecules. DC’s especially mature ones express high levels of both MHC-I and MHC-II molecules.

1.2.2.1. MHC-I restricted antigen presentation (Endogenous pathway) MHC-I molecules present endogenously synthesized peptides (synthesized by APC itself) of either self or pathogen origin (Fig. 1.10). The proteins are degraded in cytosol into peptides by proteasome and then transported into endoplasmic reticulum through the transporters of antigen-processing (TAP) molecules where they are loaded onto MHC-I molecules. This MHC I-peptide complex is then expressed on the surface of DC and the antigen is presented to CD8+ T cells (Heath and Carbone, 2001; Villadangos and Schnorrer, 2007).

1.2.2.2. MHC-II restricted antigen presentation (Exogenous pathway) In contrast to the endogenous pathway, MHC-II molecules present to CD4+ T cells, the peptides derived from the proteins that enter the cell through the endocytosis. The antigens are endocytosed either by pinocytosis, by phagocytosis or by receptor-mediated endocytosis. The proteins are degraded in the endosome by cathepsins (proteases localized in lysosomes) and

Introduction other hydrolytic enzymes (Neefjes et al., 1990; Wilson and Villadangos, 2005). MHC-II molecules from endoplasmic reticulum move through the Golgi apparatus into the vesicle containing the degraded proteins. The peptide fragments are loaded onto the MHC-II molecules and presented on the cell surface to CD4+ T cells (Heath and Carbone, 2001; Villadangos and Schnorrer, 2007) (Fig. 1.10).

Fig 1.10: Antigen presentation pathways in dendritic cells. Adapted from (Villadangos and Schnorrer, 2007).

1.2.2.3. Cross-presentation DCs have a unique ability to present endocytosed antigens on MHC-I molecules to CD8+ T cells through a mechanism termed as cross-presentation or cross-priming (Bevan, 1976; Joffre et al., 2012) (Fig. 1.10). The capability to cross-present antigens enables DCs to play vital roles in induction of tolerance in immunity against viruses and tumors (Heath et al., 2004). APC starts with the exogenous pathway but diverts the antigens to endogenous pathway via cytosolic diversion. By this way the APC circumvents the endogenous pathway which involve the synthesis of antigens after infection of the cell. So, DCs can present antigens to CD8+ T

Introduction cells without being infected (Heath and Carbone, 2001; Villadangos and Schnorrer, 2007). The underlying mechanisms behind cross-presentation still remain to be defined (Rock and Shen, 2005; Villadangos and Schnorrer, 2007).

1.3. Friend Virus In the current search for HIV vaccines, the absence of good animal models is a major limitation. Beside humans, chimpanzees are the only species that can be reproducibly infected with HIV-1. However, these animals do not progress to AIDS and can usually control the virus. Further, chimpanzees are very expensive to work with and various ethical concerns create a hindrance in the way of large scale HIV vaccine experiments. A substitute to this model is macaques, which could be infected with Simian Immunodeficiency virus (SIV) or chimeric SIV/HIV viruses known as SHIVs. However, there are a number of factors which have limited the widespread utility of this model, for instance, high costs, restricted number of monkeys available, elaborate infrastructure involved, limited knowledge about immunology of monkeys, long follow-up period, and differences in the pathology compared to human HIV-infections may limit the interpretations of the results.

Mouse models on the other hand, have an advantage over other animal species in terms of elucidating fundamental concepts in immunology. This is due to the fact that mouse models are easier to handle, different immunological manipulations can be employed and mice with different genetic backgrounds can be used (Halemano et al., 2013). The serious disadvantage is that both HIV and SIV cannot infect mice and hence other oncogenic murine retroviruses need to be used. Still, most of the oncogenic murine retroviruses cannot induce a fatal infection in adult immune-competent mice (Dittmer and Hasenkrug, 2001). An option is to genetically “knock-in” human genes in mice to enhance HIV-1 replication, but due to the presence of several restriction blocks this approach proved to be quite challenging (Zhang et al., 2008). An alternative way out could be engrafting a human immune system in immune- compromised mice (Denton and Garcia, 2009). However, currently several factors limit their full utility for immunological studies, such as, high costs, immaturity of the adaptive immune response, inconsistency in engraftment and failure to completely “knock-out” specific genes. Therefore, such retroviruses which may cause disease in “normal” mice could be the best option as a starting point for basic proof-of-concept immunological approaches and for basic vaccine discovery. Friend Virus (FV) is one choice which is useful for studying the basic immunological mechanisms in both acute and chronic retroviral infections (Dittmer and Hasenkrug, 2001; Halemano et al., 2013).

Introduction

1.3.1. Friend Virus as a platform to investigate immune response in retroviral infections In 1957, Charlotte Friend, an American virologist, discovered a novel retroviral disease in mice presenting spleen expansion, erythroleukemia and death (Friend, 1957). This disease known as Friend disease has proved to be a powerful tool for investigating the multistage carcinogenesis (Ney and D'Andrea, 2000). Friend Virus is regarded as one of the most well- characterized model of retroviral infections in healthy mice (Halemano et al., 2013). FV is a retroviral complex which consists of two components: the replication competent, non- pathogenic component helper virus, Friend Murine Leukemia Virus (FMuLV); and the pathogenic, replication-defective Spleen Focus-forming Virus (SFFV). SFFV cannot replicate on its own that is why it is produced in cells co-infected by FMuLV and it spreads by being enveloped in FMuLV encoded particles to carry its genome.

1.3.2. Friend virus induced erythroleukemia Pathology in FV infected sensitive mice is characterized in the first stage by chronic viremia, polyclonal proliferation of erythroid precursor cells leading to immense splenomegaly within 2 weeks; and progressing to the second stage of disease causing erythroleukemia and death in one to two months subsequent to the infection (Fig. 1.11).

Fig 1.11: FV induced multistage erythroleukemia in mice. Modified from (Cmarik and Ruscetti, 2010).

Introduction

The cause of the splenomegaly is the false proliferative signal induced by the association of SFFV altered form of envelope (Env) glycoproteins, gp55, to the erythropoietin receptors (EpoR) on erythroid cells (Fig. 1.12). This is how infection produces a prominently expanded population of actively dividing cells vulnerable to FV infection. The mice that express an immune response with adequate speed and potency to prevent accumulation of these two transformation-associated events are referred to as resistant strains. Nevertheless, even the most resistant strains are never able to entirely eradicate the virus-infected cells (Halemano et al., 2013; Hasenkrug and Dittmer, 2007). The highest viral loads in FV infected mice are existing in spleen and the bone marrow (Littwitz-Salomon et al., 2017).

Fig 1.12: SFFV envelope protein vs polytropic MuLV envelope protein. The precursor Env of a typical polytropic MuLV is cleaved into SU and TM proteins. On the other hand, SFFV Env glycoprotein comprises of fused SU and TM sequences, has a unique transmembrane region and lacks an intracellular extension due to a large deletion that eradicates the cleavage site between SU and TM, as well as insertion of a single base pair in TM. Adapted from (Cmarik and Ruscetti, 2010).

1.3.3. Advantages of FV as retroviral infection model Beside all limitations, the advantages of FV model to study the immunological responses upon retrovirus infection include the ability to work in genetically defined mice, for instance, 20

Introduction transgenic, congenic and knock-out strains; accessibility to a wide range of immunological reagents; and similarity between several features of FV and HIV infections and the resultant immune responses. Therefore, this mouse model offers an opportunity to explain the basic concepts regarding retroviral infection immune responses and helps to identify factors involved in chronic retroviral infections as discussed in more detail below (Hasenkrug and Dittmer, 2000).

1.3.4. Comparison of FV and HIV-1 FV (gammaretrovirus) and HIV-1 both belong to the family Retroviridae. The best studied retrovirus is HIV-1, which is a lentivirus. In contrast to HIV-1, FV is a simple retrovirus which encodes for Gag, Pol and Env, which encode the proteins that are assembled into the new virus particles. HIV-1 encodes for multiple accessory genes in addition to Gag, Pol and Env (Fig. 1.13).

Fig. 1.13: Major structural genes of retroviruses (gag, pol, env), and comparatively complex lentiviral genomes due to the accessory genes, such as rev, tat, vif, vpu, nef, vpr. Adapted from (Power, 2001).

The two viruses also differ in that HIV-1 infected cell usually die rapidly (within a few days) compared to the FV infected cells (Rein, 2011).

The comparison and contrast between FV and HIV-1 is summarized in Table 1.3. Although FV and HIV-1 are different retroviruses, FV as a retrovirus model is very helpful in providing

Introduction basic information on retrovirus-specific cell mediated immune responses (Halemano et al., 2013).

Table 1.3: FV vs HIV-1. Modified from (Halemano et al., 2013)

Feature FV HIV-1

Family Gammaretrovirus Lentivirus

Host Mice Humans, chimpanzees

Major route of Not known Mucosal, intravenous infection

Target organs Bone marrow, lymphoid organs Lymphoid organs, CNS

Target cells Erythroblasts, B cells CD4+ T cells, macrophages

Target cell receptors Mouse cationic amino acid CD4, CCR5, CXCR4 transporter 1

Viral entry protein Env gp85: gp70, SU; p15E, TM Env gp160: gp120, SU; gp41,TM

Pathogenesis Erythroblasts proliferation CD4+ T cell exhaustion Disease Splenomegaly, erythroleukemia AIDS

1.3.5. Morphology of FV The typical FV structure is enveloped, roughly spherical with a diameter of around 100- 120nm (Yeager et al., 1998). As shown in Fig. 1.14, the sphere is surrounded by a lipid bilayer derivative of the membrane of the virus-producing cell. The retroviral envelope plays three distinct roles including the protection from the extracellular environment, assisting in the entry and exit from the host cells via endosomal membrane trafficking, and facilitating the straightforward entry into the cells by fusing with their membranes (Saxena and Chitti, 2016).

The N-terminal part i.e., matrix (MA) of the Gag (group-specific antigen protein) molecules is in contact with the lipid bilayer and its C-terminal nucleocapsid (NC) domains protrude into the inner side of the particle, probably in contact with RNA. The virus particles also hold ∼1– 300 Gag-Pol (Gag-polymerase) polyprotein molecules, in which Gag extends at its C- terminus by reverse transcriptase (RT), protease (PR), and integrase (IN). The trimers of the

Introduction envelope (Env) protein span the membrane, with the gp70 surface glycoprotein (SU) on the external side of the particle, bound to the p15E transmembrane (TM) protein (Rein, 2011).

Fig. 1.14: A retroviral particle (http://theses.ulaval.ca/archimede/fichiers/23658/ch02.html).

1.3.5.1. Replication cycle of FV The infection initiates when mature and infectious virion carrying an SU glycoprotein on its surface binds to a receptor on the exterior of the host cell. Several stages of the life cycle of retrovirus (Fig. 1.15) are as follows:

Stage 1: Infection initiates by the viral Env protein-induced binding of the mature virion to the host cell surface receptor.

Stage 2: The mature core is cleaved within the cytoplasm.

Stage 3: Dimeric RNA of virus is copied into double-stranded DNA.

Stage 4: The DNA copy is transported into the nucleus (possibly during mitosis when the nuclear membrane disrupts) and is inserted into the chromosomal DNA of the infected cell.

Stage 5: Transcription of the DNA followed by the transfer of the RNA product from nucleus to the cytoplasm.

Introduction

Stage 6: In the cytoplasm some molecules are translated into viral structural and regulatory proteins, while the others are encapsulated into new virions; subsequently permitting viral assembly and eventual budding of progeny virus particles.

Stage 7: Immature virions are released from the infected cell.

Stage 8: Eventually, protease in the immature virions cleaves the viral precursor polyproteins, transforming immature into mature, infectious virions (Goff, 2007; Rein, 2011; Zhang et al., 2015).

Fig. 1.15: Stages in replication cycle of retrovirus. RTC: reverse transcription complex, PIC: pre-integration complex. Adapted from (Goff, 2007).

1.3.5.2. Maturation of FV virion The virus particle releases from the infected cell in the form of an “immature particle”, which carries thousands of rod-shaped Gag molecules are arranged, in an unfinished or imperfect hexameric lattice. The particle undergoes maturation once released from the cell (Fig. 1.16). Proteolysis of Gag by Protease results in four cleavage products, i.e., matrix (MA), p12, capsid (CA), and nucleocapsid (NC). The Pol domain of Gag-Pol is also lysed leading to the 24

Introduction release of free Protease (PR), reverse transcriptase (RT), and integrase (IN) proteins. The C- terminal 16 residues of TM are removed, resulting in the mature TM protein p15E (in literature sometimes this shorter protein is termed as p12E). This breakdown in Gag leads to a major transformation in the overall architecture of the virion, with CA molecules rearranging in the interior of the particle into a polygonal to nearly spherical assembly, the “mature core or capsid” of the particle. This mature core encloses a complex of the viral RNA with NC protein; RT and IN are also supposed to be inside this assembly (Machinaga et al., 2016; Rein, 2011).

Fig. 1.16: Morphology of immature and mature FMuLV particle (http://viralzone.expasy.org/all_by_protein/67.html).

1.3.5.3. Genome of FV FV genome encodes only for the three polyproteins which make up the progeny virus particles. It has a positive-sense RNA genome containing a 5′ LTR, 5′ leader sequence, gag, pol, env, and a 3′ LTR. The Pol proteins are primarily synthesized together with Gag, in a large Gag-Pol fusion polyprotein. Gag and Gag-Pol are translated from full- length viral RNA, identical in sequence to the genomic RNA existing in the virion. Possibly the Gag-Pol polyprotein is fused into assembling virions due to “coassembly” of its Gag moiety with Gag polyproteins.

Introduction

The Env protein of FV is translated from a singly spliced mRNA. There exists an overlap of 58 bases between the beginning of the Env coding region and the end of the Pol coding region (Rein, 2011).

1.3.5.4. Proteins of FV Proteins which are required for the inner assembly of the virion, i.e., the matrix, capsid, and nucleocapsid proteins, are encoded by the gag region. The enzymatic proteins, i.e., the reverse transcriptase, protease, integrase, and RNase, are encoded by the pol region. The Env protein which is encoded by the env region is projecting out from the viral surface. The Env protein is synthesized as a precursor protein (gpr85), which is later divided into two subunits, the surface (SU) and transmembrane (TM) proteins (Machinaga et al., 2016).

1.3.5.4.1. Gag In principle, the orthoretrovirus particle is assembled by association of Gag protein molecules. Orthoretroviral Gag proteins comprises of three domains, which generate three distinct proteins in the mature particle (Fig. 1.17). The first domain, MA domain at the N-terminus of Gag is required for directing the protein to the plasma membrane of the virus-generating cell.

The second domain, CA is responsible for most of the interactions between Gag molecules resulting in the assemblage of the immature virion. The CA molecules reassemble into the mature core once they are released from the Gag polyprotein by PR.

The third domain, NC domain is principally responsible for the interactions of Gag proteins with RNAs. During the infection, free NC protein acts as a vital cofactor in reverse transcription.

Figure 1.17: Gag protein from FV. Adapted from (Rein, 2011).

FMuLV also encodes a further form of the Gag polyprotein, termed as “glyco-Gag” or gPr80Gag. This protein varies in sequence from the “standard” Gag in that it is extended on the N-terminal (Fig. 1.18). Comparatively little glyco-Gag is incorporated into virions which is expressed at the plasma membrane of infected cells (Fujisawa et al., 2001). 26

Introduction

The functional importance of glyco-Gag is still not clear. Initial studies showed that it is required for effective replication and pathogenicity in mice but is not vital for replication of Murine Leukemia Virus in cell culture (Corbin et al., 1994; Schwartzberg et al., 1983). Later in a study it was described that the precise assembly of standard Murine Leukemia Virus Gag into spherical immature particles in cell cultures is compromised in the absence of glyco-Gag (Low et al., 2007).

Glyco-Gag Gag

Fig 1.18: Glyco-Gag and Gag. Adapted from (Low et al., 2007).

1.3.5.4.2. Pol The cleavage products of the Gag-Pol polyprotein comprise of protease (PR), reverse transcriptase (RT), and integrase (IN). PR catalyzes the cleavages resulting in viral maturation.

RT during infection reverse transcribes the viral genome leading to the synthesis of its DNA copy. This function includes three distinct enzymatic activities: synthesis of RNA-templated DNA, synthesis of DNA-templated DNA, and cleavage of RNA strand in an RNA-DNA hybrid, eradicating the RNA template right after synthesis of the complementary DNA strand.

The integration of retroviral DNA into the host genome is catalyzed by the viral IN protein (Guan et al., 2017). IN enzyme is responsible for two catalytic activities: first is “3′ end processing”, in which IN eliminates two nucleotides from the 3′ end of each strand of the DNA to be integrated; and the second activity is “strand transfer”, wherein the new 3′ ends are incorporated into chromosomal DNA in the host cell (Rein, 2011).

1.3.5.4.3. Env Env protein is synthesized as a precursor protein in the rough endoplasmic reticulum and then in Golgi apparatus it is glycosylated. It becomes cleaved in the Golgi by a cellular protease 27

Introduction into two products, the large, N-terminal surface glycoprotein (gp70SU) and the C-terminal transmembrane protein (p15ETM) (Fig. 1.19). A trimer of these SU-TM complexes is then transferred to the cell surface. During virus maturation it undergoes an additional cleavage: PR eliminates the C-terminal 16 residues, also termed as the “R peptide”, from the cytoplasmic tail of the TM protein (Rein, 2011).

The role of the Env protein is to enable fusion between the virus membrane and the membrane of the host cell. The cleavage between SU and TM is certainly required for the function of Env (Freed and Risser, 1987). Possibly, this cleavage is important because it places the fusion peptide at the N-terminus of TM instead of in the interior of the Env polyprotein (Rein, 2011). The importance of Env was also demonstrated in study where it was shown that only the mice immunized with a vaccine expressing the entire FMuLV (helper virus) envelope protein were completely protected against FV-induced erythroleukemia (Hasenkrug et al., 1998b).

Fig 1.19: Illustration of Env protein of FV. Env protein comprises of a complex of gp70SU and p15ETM. The gp70 consists of two domains, at its N-terminus there is Receptor binding domain (RBD) and at its C-terminus it has “C-term”, which are separated by a proline-rich linker. P15E comprises of, from N- to C-terminus, the fusion peptide (FP), an N- terminal helical domain (helix A), a short C-terminal helical domain (helix B), and a C- terminal domain (brown) which spans the viral membrane (yellow). Gp70 is outside the virus particle and is linked to p15E by a disulfide bond between a cysteine in a (Cont. on next page)

Introduction

CXXC moiety within its C-terminal domain and the last cysteine in a CX6CC moiety in p15E. Adapted from (Rein, 2011).

1.3.6. Pathology of Friend disease: EpoR and gp55 Molecular interactions Gp55 is the truncated form of Env glycoproteins in SFFV which interacts with the erythropoietin receptors (EpoR) at the cell surface as well as inside the endoplasmic reticulum of the infected erythroblasts. This interaction of gp55 leads to the stabilization and activation of the EpoR (Yoshimura et al., 1990). Gp55 does not activate the human EpoR but selectively stimulates the murine EpoR, in spite of the fact that these two receptors have 92% amino acid identity (Showers et al., 1993). The critical binding between the murine EpoR and gp55 takes place within the transmembrane area of these cell surface proteins (Chung et al., 1989; Constantinescu et al., 1999), and a productive contact between gp55 and the EpoR occurs when expression of both proteins takes place at the cell surface (Li et al., 1995; Zon et al., 1992). It is not possible to trigger the gp55-EpoR interaction by cell-to-cell contact or by means of administrating soluble gp55 to EpoR-expressing cells (Ney and D'Andrea, 2000).

Fig. 1.20: Illustration of Multistage Friend erythroleukemia disease Model resulting from FV infection (adapted from (Ney and D'Andrea, 2000). 29

Introduction

The first stage of the FV infection comprises of SFFV provirus incorporation into the host genome and encoding of oncogenic envelope protein, gp55, which complexes with the EpoR and possibly also SF-Stk (short form of stem-cell-kinase receptor), both at the cell surface and within an intracellular compartment. This complex formation results in the constitutive activation of the EpoR. SF-Stk may play its role in this interaction mechanism in one or several ways. Such as, SF-Stk may improve the affinity of EpoR-gp55 interaction, up-regulate the expression of cell surface EpoR, act as a co-receptor that triggers additional downstream signaling events, or activate exclusive downstream signaling processes that are required for erythroblast transformation (Fig. 1.20 A) (Ney and D'Andrea, 2000).

The second stage of the disease is characterized by the up-regulation of Sfpi1/PU.1 (spleen focus forming virus proviral integration 1) expression due to the proviral integrations and inactivation of wild-type p53 (tumor suppressor gene) along with other events (Starck et al., 1999) . Even though this is only hypothetical, Sfpi1/PU.1 perhaps causes the activation of SF- Stk as well as other target genes, leading to the development of erythroleukemia (Fig. 1.20 B) (Ney and D'Andrea, 2000).

1.3.7. Resistance to FV infection: role of SF-Stk Not all mice are susceptible to FV-induced disease. Animals, such as C57BL/6, C57BL/10, C57BR and C58 mice do not show the signs of erythroleukemia and ultimate death which is in contrast to the FV- induced disease sensitive mice, for instance DBA/2, C3H, AKR, NZB, BALB/c and SJL/J strains (Hasenkrug, 1999; Kumar et al., 1974; Pincus et al., 1971; Raikow et al., 1985; Silver and Fredrickson, 1983). One of the possible underlying reasons for this resistance could be the structural difference of Stk between differently susceptible mice strains (Fig. 1.21). Stk belongs to a vast family of cell surface tyrosine kinase receptors. SF- Stk is deficient in the extracellular ligand binding domain of Stk while it still carries the transmembrane and the tyrosine kinase domains (Iwama et al., 1994). Fv2 gene (Friend virus susceptibility gene 2) encodes the Stk. Fv2 does not interfere with the retroviral life cycle or its entry into the cells, rather Fv2 defines the capability of FV-infected erythroblasts to proliferate in response to gp55 (Bondurant et al., 1985). Internal promoter within the Stk gene naturally initiates the transcription of SF-Stk. The mice resistant to Friend virus lack this internal promoter (Persons et al., 1999). In FV-infected mice, gp55, EpoR and SF-Stk are effectors in signaling pathways including STATs, PI3-kinase/AKT, Grb2/Gab2, the Lyn kinase, the p38MAP kinase, and the ERK1/2 MAP kinases. Stimulation of these signaling pathways by the gp55-EpoR and gp55-sf-Stk complexes results in the dysregulation of

Introduction proliferation, differentiation and survival of FV-infected susceptible mice leading to acute erythroblastosis. The expression of Sf-Stk influences the vulnerability to gp-55 induced erythroblastosis (Moreau-Gachelin, 2008). Results from several experiments indicate that Fv2 resistance does not independently protects against FV-induced erythroleukemia but works in collaboration with immune system by limiting the acute infection for sufficient period to let the FV-specific immunity to develop and help in the recovery (Hasenkrug, 1999).

It has been reported in the studies that a direct interaction between SF-Stk and EpoR could be the underlying mechanism. The constitutive expression of SF-Stk can play a role in the immortalization of FV- infected erythroblasts. It has been shown in SF-Stk-deficient mice, for instance in the C57BL/6 mice, that they almost do not lose their normal erythropoietic response; while, gp55-regulated erythroid proliferation is reduced. Consequently, they express resistance against Friend virus–induced erythroleukemia (Cmarik and Ruscetti, 2010; Ney and D'Andrea, 2000).

Fig 1.21: Comparison of full length and short form-Stk. The full length Stk, a tyrosine kinase acts as the cell surface receptor for macrophage stimulating protein (MSP). The short form of Stk (sf-Stk), lacks the MSP binding domain. Mice strains resistant to FV-induced erythroleukemia do not encode sf-Stk owing to a 3 bp deletion in the internal promoter. Adapted from (Cmarik and Ruscetti, 2010).

Introduction

1.3.8. Immune responses in FV infection Various studies have described substantial connections between recovery from FV erythroleukemia and several parameters of the immune response in FV-infection. Succeeding research has proven that each of these responses is not only correlated but is also required for recovery. Spontaneous recovery from FV infection in genetically resistant mice requires immunological support from all the effector arms of the immune system, including antibodies and B cells (Chesebro et al., 1990; Hasenkrug and Chesebro, 1997), CD4+ T cells (Robertson et al., 1992), CD8+ T cells (Hasenkrug et al., 1995; Hasenkrug and Dittmer, 2000) and Natural killer T cells (NKT) (Littwitz-Salomon et al., 2017).

1.3.8.1. Role of CD8+ T cells in FV-specific immunity FV infection is characterized by rapid onset of splenomegaly and viremia in around 4-5 days post infection in the acute phase (Bubbers and Lilly, 1977) then reduces rapidly as soon as the immune response develops and achieves control over the acute phase of infection (Robertson et al., 1992) (Fig. 1.22). CD8+ T cells are vital for the recovery from the acute phase of FV infection and even the resistant mice, for instance, C57BL/6 mice are not able to control acute infections in the absence of a CD8+ T cell response as shown by the depletion experiments (Hasenkrug, 1999). CD8+ T cell responses correlate with a reduction in spleen size of FV- infected animals and the protection is regulated by IFN-γ and three critical components of cytotoxic granules, perforin, granzymes A and B which eliminate virus-infected cells by apoptosis of target cells (Zelinskyy et al., 2005). In FV infection, the necessity for one or more of these cytotoxic granules is dependent upon the phase, acute or chronic, of infection.

On contrary, CD8+ T cells seem to have no effect on the control of chronic infection as indicated by depletion experiments (Robertson et al., 1992). Even though, FV-specific CD8+ T cells express activation markers in persistently infected mice, they are functionally impaired to produce the cytolytic granules and of IFN-γ. Moreover, CTL were also shown to be not effective in mounting cytolytic activity in vivo at later stages of infection (Hasenkrug and Dittmer, 2007; Zelinskyy et al., 2005).

1.3.8.2. Role of CD4+ Regulatory T cells in FV-specific immunity The CD8+ T cells which effectively respond in the acute phase of FV infection are rendered functionless during chronic phase of the infection. A reason for this transition could be any form of immunological escape from CD8+ T cells which maybe mediated by poor recognition of infected cells. However, FV-specific CD8+ T cells from FV-specific CD8 TCR transgenic mice when adoptively transferred to chronically infected mice effectively recognized the 32

Introduction antigen and underwent activation and proliferation. Though, these transferred CD8+ T cells were only able to reduce viral loads in acutely infected animals but not in persistently infected mice (Hasenkrug and Dittmer, 2007). It was described that CD4+ T regulatory cells (Tregs) were responsible for the suppression of CD8 function during chronic FV infection (Iwashiro et al., 2001). The immunosuppressive action is mediated by Treg cells during persistent infection by inducing dysfunction in CD8+ T cells (Dittmer et al., 2004), but it did not affect the antigen recognition and proliferation of CD8+ T cells (Li et al., 2008).

Fig. 1.22: T cell responses during Friend retrovirus infection. Adapted from (Hasenkrug and Dittmer, 2007).

1.3.8.3. Role of CD4+ Helper T cells in FV-specific immunity CD4+ T cells are classically termed as helper T cells whose main function is to provide help for immunological effectors, CTLs and B cells. However, their subpopulations have shown antiviral functions in viral infections (Hasenkrug and Dittmer, 2007).

Introduction

In contrast to CD8+ T cells, CD4+ T cells are likely to be important for controlling FV replication during chronic phase of infection. Adaptive depletion of CD4+ T cells in persistently infected mice resulted in the reactivation of infection (Robertson et al., 1992). Chronically FV-infected mice with CD4+ T cells depletion demonstrated significantly higher viral loads in spleen compared to no-depleted mouse strains (Hasenkrug et al., 1998a). Contact dependent cytotoxicity and secretion of IFN-γ are both responsible for the reduction of virus replication (Hasenkrug and Dittmer, 2007).

1.3.8.4. Role of antibodies and B cells in FV-specific immunity Another factor required for recovery from FV infection is virus neutralizing antibodies, their production is controlled by non-MHC gene, Rfv-3 (Hasenkrug and Chesebro, 1997). FV infection increases mortality more than 90% in mice which fail to mount a virus-neutralizing antibody response (Hasenkrug et al., 1995). B cells are important not only for antibody production but they also play pivotal role in antigen presentation and cytokine production. Depletion of B cells significantly diminished both CD8+ and CD4+ T cell responses to FV infection (Schultz et al., 1990).

1.3.8.5. Role of Natural Killer T (NKT) cells in FV-specific immunity NKT cells are innate-like T lymphocytes which are recognizing glycolipid antigens presented by MHC class I-like molecule CD1d. NKT cells are able to secrete various kinds of Th1 and Th2 cytokines which results in immune response or immune suppression. In a recent study with FV-infected C57BL/6 mice a significantly higher number of anti-inflammatory cytokines, IL-10 as well as IL-13, producing NKT cells was observed 3days post infection (acute infection). Whereas, there was no increase observed in the pro-inflammatory cytokines, i.e., INF- and TNF-α (Littwitz-Salomon et al., 2017).

In addition to anti-inflammatory cytokines production, NKT cells also showed an enhancement in the percentage of degranulation marker CD107a (lysosomal-associated- membrane protein-1 [LAMP-1]) and FasL expressing NKT cells post infection. Moreover, the results from an adoptive transfer experiment of NKT cells from FV-infected mice into acutely FV-infected mice, showed a significant decrease in viral load in vivo post transfer (Littwitz- Salomon et al., 2017).

To summarize, CD8+T cells play role in the recovery from acute FV infection. At later stage of infection the effector function of CD8+T cells is impaired by Tregs. At this point, CD4+ T cells take over the control of viral replication. However, the recovery from persistent FV

Introduction infections is not observed, and animals stay chronically infected. Studies suggest that efficient CD8+ T cell, CD4+ T cell and B cell responses collectively are critical for the effective control of the retroviral infections. Therefore, future efforts for the generation of immunotherapy or vaccine against retroviruses, HIV for instance, should take into account the induction of multiple arms of the immune system.

Aims of the Study

2. AIMS OF THE STUDY

DCs are the most potent APCs and are able to efficiently induce CD8+ T cell responses. DCs are known to express CRs on their surface, which are responsible for their ability to interact with complement opsonized viruses. The interaction of CRs and the complement-opsonized viruses might affect the infection behavior of DCs and consequently may influence the antigen presenting capacity and the induction of the CTL responses. We selected FV as a model for retroviral infections.

Our first aim was to study the relation between the opsonization of retrovirus and the infection behavior of DCs at different initial viral loads. Secondly, we aimed to dissect the role of CR3 and CR4 which are co-expressed on DCs; the ligand specificity and tissue distribution of both of these CRs share great resemblance. The specific roles of these molecules have not been distinguished in depth to date and therefore, more information is required in this direction to determine the relevance of opsonized viruses interacting with CRs. Our results may provide valuable information about the approaches to target antigens to DCs in order to develop novel and improved vaccine candidates.

Materials & Methods

3. MATERIALS AND METHODS

3.1. Materials

3.1.1. Equipment The following equipment and materials were used:

 FACS Canto II (BD, Germany)  Real time PCR, CFX-Connect Real-Time System (Bio-Rad, USA)  Thermomixer (Eppendorf, Germany)

 CO2 incubator (Memmert, Germany)  Microscope (Olympus, Japan)  Automated cell counter (Luna, Germany)  Water bath (Memmert, Germany)  Centrifuge, Rotanta 460 R (Hettich, Germany)  Millifuge (Millipore, Japan)  24-well cell culture plates; # 3524 (Corning, USA)  Polystyrene round bottom tube, 5 mL; # 352008 (BD, Germany)  Cell culture flasks, 75 cm2; # 658170 (Greiner bio-one, Austria)  Cryo vials, Cryo.S; # 123263 (Greiner bio-one, Austria)  Micro tubes, 1.5 mL; # 72.692.005 (Sarstedt, Germany)  PCR soft strips, 0.2 mL, # 710980 (Biozym, Austria)  Filter, pore size 0.45 µm; # 83.1826 (Sarstedt, Germany)  Tissue culture dishes (100×20 mm); # 353003 (BD, Germany)  Petri-dishes; # 633180 (Greiner bio-one, Austria)  Centrifuge tubes, # 227261 (Greiner bio-one, Austria)  Serological pipettes (1mL), # 604181 (Greiner bio-one, Austria)  Serological pipettes (10mL), # 607180 (Greiner bio-one, Austria)  Serological pipettes (25mL), # 760180 (Greiner bio-one, Austria)  Filter tips, DNase-, RNase- and Pyrogen-free (Biozym, Austria)  Needle: 25G × 1", # 9186158 (Braun, Germany)  Syringe: 1mL Injekt-F, # 9166017V (Braun, Germany)  iScript One-Step RT-PCR Kit; # 1708895 (Quanta-bio, Germany)

Materials & Methods

3.1.2. Chemicals and media The following chemicals and media were used in the study:

 RPMI 1640 without L-Glutamine; # 31870-025 (Gibco, USA)  RPMI 1640 with L-Glutamine; # BE12-702F (Lonza, Belgium)  PBS without Ca & Mg; # BE17-516F (Lonza, Belgium)  Fetal Bovine Serum (heat inactivated); # 10500-064 (Gibco, USA)  Lympholyte; # CL5035 (Cedarlane, Canada)  Accutase; # 423201 (BioLegend, USA)  Polybrene (Sequabrene); # S-2667 (Sigma, USA)  Dimethyl sulfoxide, DMSO; # D2650 (Sigma, USA)  Bovine Serum Albumin; # 8076.4 (Carl-Roth, Germany)  EDTA 0.5M; # AM9260G (Ambion, USA)  Hydrogen Peroxide, 30%; # 216763 (Sigma, USA)  Non-fat dried milk powder; # A0830 (AppliChem, Germany)  Sodium acetate, anhydrous; # 1.06268 (Merck, Germany)  Glacial acetic acid; # A02820 (AppliChem, Germany)  Ethanol, 95%; # 1.00983 (Merck, Germany)  Formaldehyde, 37%; # 7398.1 (Carl-Roth, Germany)  GM-CSF; # 554586 (BD, Germany)  IL-4; # 554586 (BD, Germany)  AEC (3-Amino-9-ethylcarbazole); # A6926 (Sigma, USA)  DMF (N,N,-dimethylformamide); # 154814 (Sigma, USA)

3.1.3. Antibodies  Rabbit anti mouse IgG; # Z0259 (DAKO, Germany)  Polyclonal Goat Anti-mouse immunoglobulins HRP; # P0447 (DAKO, Germany)  Polyclonal Goat Anti-mouse immunoglobulin-FITC; # F0479 (DAKO, Germany)  Goat antiserum to mouse complement C3; # 55444 (ICN Cappel, USA)  Unless otherwise specified, all the following fluorescently labelled antibodies were purchased from Beckton Dickinson (BD), Germany: . CD3-FITC (Fluorescein isothiocyanate), CD4-FITC, CD8-FITC, CD19-FITC, CD11b-FITC, CD11c-FITC, CD103- FITC, MHC II-FITC, CD3-PE 38

Materials & Methods

(Phycoerythrin), CD4-PE, CD8-PE, CD11b-PE, CD64-PE, CD83-PE, CD86- PE, CD103-PE, Ly6C-PE (eBioScience, USA), Ly6G-PE, Cd16/CD32-PE, CD21/CD35-PE, CD4-PerCP-Cy5.5 (Peridinin-chlorophyll-protein complex), CD8- PerCP-Cy5.5, CD19- PerCP-Cy5.5, CD11b- PerCP-Cy5.5, Dec205- PerCP-Cy5.5 (BioLegend, USA), Ly6C-PE Cy7 (Phycoerythrin – Cy7), F4/80- PE Cy7 (eBioScience, USA), CD4-APC (Allophycocyanin), CD8-APC, CD11c-APC, CD86-APC, CD21/CD35-APC, CD19-APC Cy7, CD3-V450, Cd11b-V450, CD11c-V450.

3.1.4. Buffers and solutions

Description Composition

RPMIØ RPMI 1640 without L-Glutamine; # 31870-025, Gibco

RPMI+ RPMI 1640 with L-Glutamine; # BE12-702F, Lonza 10% FCS

FACS buffer PBS 2% FCS 5mM EDTA

FACS-Fix PBS 1% Formalin

Freezing medium FCS 10% DMSO

AEC solution AEC tablet was dissolved in 2.5 mL of DMF (8 mg/mL) and then diluted to 50 mL with 50mM acetate buffer, pH 5.0. The

reaction was started by adding 25 µL of 30% H2O2 (0.5µL/mL).

Materials & Methods

3.1.5. Animals C57BL/6 (B6) mice were bred and maintained in house at our animal facility. FV-specific CD8+ TCR transgenic (tg) mice carrying a TCR transgene which identifies the gag peptide of FV, were provided by Prof. Ulf Dittmer, Institute of Virology, University of Duisburg, Essen, Germany. CD11b-/- and CD11c-/- mice having the background of C57BL/6 mice were received from Dr. Adman Verschoor, University of Lübeck, Germany.

3.2. Methods

3.2.1. Mice Experiments were carried out with 3 to 6 month old C57BL/6 mice, CD11b-/- and CD11c-/- mice, and FV-specific CD8+ TCR transgenic (tg) mice which carry a TCR transgene encoding for TCR which identifies the gag peptide of FV (Chen et al., 1996; Ejaz et al., 2012). All animals were bred and maintained in an environment free of specific pathogens in the animal facility at the Department of Hygiene, Microbiology and Social Medicine, Division of Virology, Medical University Innsbruck, Austria. They were handled according to the guidelines of the “European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes” and the Austrian law.

3.2.2. Cultivation of Mus dunni cells Mus dunni cell line is derived from the tail tissue of a female Mus dunni mouse (Lander and Chattopadhyay, 1984). The adherent Mus dunni cells were cultured in a 5% CO2 atmosphere at 37°C, in 75 cm2 cell culture flasks containing RPMI+ (RPMI 1640 + 10% FCS + 2mM L- glutamine). The cells were passaged at ~70-80% confluency. For this, the medium was removed and the cells were briefly washed with PBS. 500 µL Accutase was added and incubated at 37°C for 2-3 min to detach the adherent cells. 9.5 mL of fresh RPMI+ was added to the cell culture flask. 500 µL of this cell suspension was transferred to a fresh cell culture flask already containing 9.5 mL of fresh medium. The cells were split twice a week.

3.2.2.1. Storage of Mus dunni cells For longer storage, 1×106cells were suspended in per mL of the freezing medium. The suspension was aliquoted in 1 mL portions into freezing tubes and frozen overnight at -80°C. Later, the tubes were transferred to liquid nitrogen. Thawing of cells was carried out in a water bath at 37°C. Cells were washed with PBS to remove DMSO, resuspended in 10 mL of RPMI+ and then transferred to cell culture flasks.

Materials & Methods

3.2.3. Virus stocks production FMuLV and fluorescently-labelled FMuLV-Wasabi (wFMuLV) stocks for in-vitro infection experiments were produced in susceptible Mus dunni cells. 1×106 cells were seeded in 75 cm2 cell culture flask in 10 mL of RPMI+ and incubated at 37°C overnight. Cells pretreated with 8 µg/mL polybrene were infected with multiplicity of infection (MOI) 5 of F-MuLV. After two hours of incubation at 37°C, fresh medium was added to make the total volume up to 10 mL. The input virus was removed 48 hpi by washing the cells twice with fresh medium. The supernatants (SNs) containing virus were collected each 24 hours and the cells were replenished with fresh medium. SNs were centrifuged to remove cell debris, aliquoted and stored at -80°C until further use. The virus stocks were free from lactate dehydrogenase- elevating virus (LDV).

The fluorescent virus (wFMuLV) with incorporated green florescent protein (GFP), wasabi, was provided by Prof. Ulf Dittmer, Institute of Virology, University of Duisburg, Essen, Germany. The fluorochrome is incorporated in the envelope of FMuLV; therefore, not only the virus but also the infected cells produce detectable green fluorescence.

Fig. 3.1: Uninfected M. dunni cells (A). M. dunni cells infected with MLV 3 dpi (B). Adapted from (Jung et al., 2004).

3.2.4. Quantification of virus by infectious centers (IC) assay We used a method described by (Robertson et al., 1991) to estimate the viral loads of FMuLV. In this assay host cell monolayers are infected with several dilutions of the virus

Materials & Methods sample and allowed to incubate for a certain period, creating localized clusters (foci) of infected cells. The number of spots (infectious centers) corresponds to the number of focus forming virus units per mL (FFU/mL). 2.5 × 104 cells/well of Mus Dunni cells in 500 µL of RPMI+ were seeded in 24-well plate and incubated at 37°C overnight. The cells were co- cultured with serial four-fold dilutions of virus-containing SNs (either from infected dunni cells or bmDCs), total volume per well was made up to 2 mL. Polybrene (8µg/mL) was added to facilitate the binding of virus to the cell surface. The cells were co-incubated for three days at 37°C. The replicated virus is transferred from infected dunni cells to the daughter cells. Virus focuses were formed; each focus representing one infected cell. After the incubation period the medium was removed and the cell were fixed with 95% ethanol for 5 min and washed with PBS containing 0.1% BSA. The fixed cells were incubated with FMuLV envelope-specific monoclonal antibody 720 for 1 hr. at room temp. Plates were washed twice with PBS containing 0.1% BSA and stained with goat anti-mouse antibody conjugated with horse-radish peroxidase for 30 min. Subsequently, the plates were washed thrice with PBS containing 0.1% BSA and developed with freshly prepared solution of AEC for 20 min. The developed red spots represented the virus foci which were counted after washing and the viral loads were back calculated.

3.2.5. Flow cytometry Flow cytometry is used to differentiate and count cells and particles which are labelled with fluorescent antibodies. Fluorescence activated cell sorter (FACS), was used for flow cytometric analyses. 1x106 cells were washed in FACS buffer and the pellet was resuspended in 50 µL of FACS buffer containing various fluorescently labelled surface antibodies (CD11b, CD11c, CD8, CD25, CD69, CD80, CD86, MHC II etc.) and incubated for 30 min at 4°C in dark. The stained cells were washed with FACS buffer resuspended in 500 µL of either FACS buffer or FACS-Fix. Propidium iodide staining was used to exclude the dead cells. Samples were measured with FACS Canto II, Becton-Dickinson, and data analyzed using FACS Diva (BD).

3.2.6. Generation of mouse bone marrow-derived DCs (bmDCs) Generation of murine bmDCs was performed by a method described previously (Inaba et al., 1992). The bones of femur and tibia were taken from C57BL/6 mouse after sacrificing it with cervical dislocation. Muscles were removed and the bones were washed with 70% ethanol, PBS and RPMI+. Both ends of the bones were cut and they were flushed with medium using 1mL syringe with a 25G needle to remove the bone marrow. The cell mass was disrupted by

Materials & Methods pipetting few times with the syringe and needle. The suspension was centrifuged resuspended in 20mL RPMI+ and carefully layered over 15mL Lympholyte-M in a 50mL centrifuge tube and centrifuged at 1800 rpm for 20min at 18°C without brakes. Centrifugation resulted in three layers with lymphocytes layer is at the interface. The uppermost layer was removed, the interface was collected carefully using 10mL serological pipette and washed twice with Wash Buffer (RPMI+ : PBS, 1:1). Subsequently, 2 × 106 bone marrow cells were cultivated in 100 mm tissue culture dish in 10 mL of DC medium [RPMI+, 10 µL recombinant mouse granulocyte-macrophage colony-stimulating factor (GM-CSF) (diluted 1:50), 10 µL recombinant mouse interleukin-4 (IL-4) (diluted 1:50)]. The cells were incubated at 37°C for three days and then supplemented with 10 mL of fresh DC medium.

On day 6 the non-adherent cells were collected in a 50mL tube, washed with RPMI+ and cultured in 20 mL fresh DC medium. On day 8 non-adherent cells in the supernatant and loosely-adherent cells were harvested and analyzed for phenotype (>80% CD11c+, >90% CD11b+, ~15% CD86+) and used for further experiments.

3.2.7. Opsonization of FMuLV The whole opsonization process was carried out on ice. Normal mouse serum (NMS) was used as a source of complement, while monoclonal antibody clone 48 was used for IgG opsonization of the virus. Medium alone served as control for virus opsonization. 400 µL FMuLV was opsonized with 80 µL of either NMS (FMuLV-C), clone 34 (FMuLV-IgG) or RPMI (FMuLV) for 60 min at 37°C. Virus was centrifuged at 13000×g for 90 min at 4°C in order to remove the opsonizing agent. The pellets were resuspended in 300 µL of RPMI each, aliquoted and stored at -80°C until further use.

3.2.8. Real-time PCR RT-PCR was applied to determine the amount of virus using FV-specific forward and reverse primers and fluorogenic probe (Metabion). Reverse transcriptase kit (iScript One-Step RT- PCR Kit) was used to carry out the reaction. The samples were analyzed in Bio-Rad CFX- Connect Real-Time System with the following run profile:

Step 1: 50°C 10 min

Step 2: 98°C 5 min

Step 3: 95°C 15 sec 60°C 30 sec 50 cycles

Step 4: 15°C hold 43

Materials & Methods

3.2.9. Virus capture assay Virus capture assay (VCA) was applied to confirm the opsonization pattern of FMuLV-C, FMuLV-IgG and FMuLV. Briefly, 96-well plate was coated using coating buffer (0.1M

NaHCO3 pH 9.6-9.8) with capturing antibody, either Goat antiserum to mouse complement C3 or Rabbit anti-mouse IgG, 50 µL each, diluted 1:10. The plate was incubated for at least 2hrs at 37°C or overnight at 4°C. Subsequently, the wells were washed with 100µl of 3% Non-fat dried milk powder in PBS and incubated with 100µL of the same for 30 min at room temp. Equal quantities of FMuLV, FMuLV-C and FMuLV-IgG as determined by RT-PCR were taken in 50 µL and incubated at 4°C overnight. Afterwards, the plates were washed thrice with 100 µl of RPMI. Lysis buffer was incorporated and samples collected for RNA isolation. RT-PCR was applied to determine the amount of captured virus.

3.2.10. In vitro productive infection of bmDCs for IC assay Day 8 bmDCs were seeded 0.5 × 106 cell/well in a 24-well plate in a final volume of 100 µL. The DCs were loaded with MOIs of 0.001–1 of differentially opsonized virus, FMuLV, FMuLV-C and FMuLV-IgG. The infectivity of virus could be influenced during the opsonization process; therefore bmDCs were loaded with equivalent quantities of viral RNA as determined by RT-PCR (Banki et al., 2010). Non-loaded bmDCs were used as control. The cells were incubated at 37°C for 2 hrs and then 400 µL of DC medium was added to the cells. Later, 24 hrs post infection (hpi), the cells were washed twice to remove the input virus, and cultured further with fresh medium. On 5 day post infection (dpi), SNs containing virus were collected, centrifuged to get rid of cells and co-cultured with dunni cells for IC assay to determine the viral concentrations.

3.2.11. In vitro infection of bmDCs for FACS analysis The infection protocol of bmDCs for these experiments was same as described above. The cells were collected at various time points, for instance, 3, 6, 12, 24, 48 or 72 hpi. Infected floating bmDCs were collected and centrifuged, while the adhered bmDCs were detached using 5mM EDTA. Both floating and adhered cells were pooled, washed with FACS buffer, stained with fluorescently labelled surface antibodies, and analyzed. The infected cells were analyzed by the expression of viral glycosylated Gag protein using a monoclonal antibody clone 34; and later incubating it with a fluorescently labelled antibody (Goat anti-mouse- FITC). The fluorescent FMuLV was detected in flow cytometer using the FITC channel.

Materials & Methods

3.2.12. Isolation of CD8+ T cells FV-specific CD8+ T cells were isolated from the spleens of FV-specific TCRtg mice. BD IMag CD8 T Lymphocyte Enrichment Set was used for the negative selection of CD8+ T lymphocytes from mouse spleen according to the manufacturer’s instructions.

3.2.13. Co-culture of FMuLV loaded bmDCs with FV-specific CD8+ TCRtg T cells To study the role of α-chain of CR3 and CR4, CD11b and CD11c respectively, in the activation the FV-specific CD8+ TCRtg T cells, co-culture experiments were carried out. For this, firstly bmDCs were isolated from C57BL/6 wild type (wt), CD11b-/- and CD11c-/- mice and on day 8 these bmDCs (1 × 106) were loaded with MOI 0.01 of differentially opsonized FMuLV. These bmDCs were washed twice 24 hpi, and then co-cultured with 1 × 106 of FV- specific CD8+ TCRtg T cells. After 24 hrs of co-culture, CD8+ T cells were stained with fluorescently labelled surface antibodies against CD25 and CD69 which are typical markers for the activation of T cells. Samples were analyzed by flow cytometry.

3.2.14. Statistical analysis The data was analyzed statistically by GraphPad Prism software. Differences between groups were performed using one-way ANOVA or Student’s t-test. P values of p<0.05 were considered as statistically significant. The data are presented as mean ± S.D.

Results

4. RESULTS

4.1. FMuLV propagation and IC assay FMuLV was successfully propagated in permissive Mus dunni cells (Fig. 4.1.1). SNs were collected every 24 hrs and were analyzed for virus concentration by IC assay. Generally the FMuLV stocks had a concentration of 106 FFU/mL.

Fig 4.1.1: Fluorescence image of Mus dunni cells infected with wFMuLV (17 dpi).

Fig 4.1.2: A 24-well plate developed for IC assay. Serial dilutions of the virus samples are used to determine the infectious titer. One red spot represents an infectious particle.

Results

Viral loads were determined by titrating virus samples on susceptible dunni cells in serial dilutions. After incubating the cultures for three days, they were fixed, stained with FMuLV envelope-specific monoclonal antibody 720 and developed with goat anti-mouse peroxidase conjugated antibody to identify foci (infectious centers). Each red spot represented a single infected cell (Fig. 4.1.2).

Several experiments were carried out to study various factors affecting the titers of the viral stocks during the cultivation process and to optimize the process of generation of the virus. The variables considered included, size of cell culture flask, number of dunni cells, initial volume of medium, MOI, the final volume of medium and the harvesting time. The highest viral concentrations could be reached when we used 75 cm2 cell culture flask, 1×106 dunni cells infected with 5 MOI of FMuLV in an initial volume of 5 mL which was made up to 10 mL 2 hpi. Harvesting the virus every 24 hrs resulted in the highest titers (Fig. 4.1.3).

Fig. 4.1.3: Pattern of FMuLV titers over several days during production process.

4.2. Loading dunni cells with FMuLV- Viability and Infection level In order to optimize the conditions for bmDCs infection experiments, we first performed the initial studies with Mus dunni cells. The cells were loaded with FMuLV in several MOIs ranging from 0.001 to 10. Non-infected cells were used as the controls. The cells were collected at pre-selected time points stretching over 3 – 72 hrs and then tested for viability of dunni cells by flow cytometry. The results did not show any significant cell death over a 47

Results period of 72 hrs, at least with the lower MOIs. After 48 hrs of infection there were signs of slight reduction in the number of living cells with higher viral loads, i.e., MOIs 1 and 10 (Fig. 4.2.1).

Fig. 4.2.1: Viability of Mus dunni cells over 72 hrs loaded with various amounts of FMuLV. Data represents the means ± S.D. of three independent experiments.

Dunni cells were also tested for the level of infection over a period of 72 hrs after loading them with various MOIs of FMuLV. As observed earlier, the level of infection increased dose-dependent with an escalation of the initial viral load. More than 90% dunni cells got infected within 48 hrs after exposure to the virus at MOI 1. Using MOI 10 infected above 90% of the cell population within the first 24 hrs of the study (Fig. 4.2.2).

Keeping the results in mind, it was decided to use highest MOI of 1 for the upcoming experiments; moreover, the viability of the infected cells was sufficiently high for at least 72 hrs during the course of experiment.

Results

Fig. 4.2.2: Infection of Mus dunni cells over 72 hrs loaded with various amounts of FMuLV. Data represents the means ± S.D. of three independent experiments.

4.3. Generation of bmDCs and their phenotypical characterization BmDCs were generated from bone marrow cells derived from C57BL/6 mice and cultivating them for 8 days in the presence of cytokines, GM-CSF and IL-4. On day 8 non-adherent DCs in the supernatant and loosely adherent DCs were collected and were characterized for their phenotype using flow cytometry (Fig. 4.3.1). Cells were analyzed for the typical DC markers, CD11c and CD11b; for maturation marker, MHC II; and for co-stimulation molecule, CD86. The FACS gating strategy was to first exclude the cell debris (Fig. 4.3.1 A) and then to gate on the singlets (Fig. 4.3.1 B). The singlets were further analyzed for CD11b and CD11c double positive cells which were normally ~85% of the total population (Fig. 4.3.1 C). The CD11c+ CD11b+ cells are believed to be comprising of two sub-populations; one represent a population that has undergone spontaneous maturation and express higher amounts of MHC II and CD86. This sub-population generally comprised of around 10 – 15% cells of the parent

Results

population. The second sub-population represent an immature phenotype of DCs expressing low or intermediate MHC II and CD86 (Fig. 4.3.1 D). In particular, the output of culture of mouse bone marrow cells with cytokines, for instance GM-CSF, is heterogeneous and comprises of granulocytes and macrophages in addition to DCs. Typical DCs are enriched in the supernatant and loosely adherent fraction of the culture and express CD11c and MHC II. On the other hand, macrophages are reported to be adherent and do not express CD11c and MHC II (Helft et al., 2015; Inaba et al., 1992). In our cultures from bone marrow, we could also identify the DC (CD11c+) and non-DC (CD11c-) fractions (Fig. 4.3.1 B).

(A) (B)

CD86 CD11c

CD11b MHC II

Fig. 4.3.1: Phenotype of representative bmDC cultures at day 8 analyzed by flow cytometry. Circles or boxes depict gates and numbers correspond to percentage of cells in each gate.

Results

4.4. Flow cytometry analysis of FMuLV-loaded bmDCs

(A) (B)

CD11c PropidiumIodide

CD86

(E) (F)

Wasabi Wasabi

CD11c CD11c

Fig. 4.4.1: FACS analysis of differentially infected sub-populations of bmDCs. Circles or boxes depict gates and numbers correspond to percentage of cells in each gate. 51

Results

To study the infection behavior of different sub-populations of bmDCs in the presence of the virus using flow cytometry, DCs were infected with fluorescent FMuLV (wFMuLV). wFMuLV is the helper virus with a fluorescent chromophore, wasabi, incorporated in its envelope. Wasabi is a monomeric GFP that can be easily detected using standard GFP filter sets. For the flow cytometric analysis, first the cell debris was excluded (Fig. 4.4.1 A), and then only the singlets were gated (Fig. 4.4.1 B). The dead cells were gated out by using propidium iodide (PI) staining; only PI negative cells were considered for analysis (Fig. 4.4.1 C). It was possible to differentiate the various sub-populations of bmDCs. The sub-population expressing high levels of CD11c and CD86 was marked as mature DC phenotype. While, cells with a high expression of CD11c but low CD86 expression were believed to be the immature phenotype of DCs. The fraction of cells which did not express CD11c was designated as non-DCs, they could be granulocytes or macrophages (Fig. 4.4.1 D). The sub- populations of DCs were then analyzed for the number of cells positive for the infection with the virus (Fig. 4.4.1 E and F). The fluorochrome wasabi is incorporated in the envelope of the virus, therefore the infected cells also produce green fluorescent signal. During FACS analysis it was possible to detect the wasabi signal in the FITC channel. The cells expressing green fluorescence were considered to be infected with the virus.

4.5. Viability and infection level of FMuLV loaded bmDCs- FACS analysis Cell death during the course of experiments may affect the infection pattern of bmDCs. Therefore, to analyze the effect of FMuLV infection on the viability of bmDCs, cells were infected with various amounts of virus ranging from MOI of 0.001 to 1. BmDCs did not show significant cell death over the period of 72 hrs, not even at the highest viral load. The findings are summarized in Fig. 4.5.1.

Results

Fig. 4.5.1: Viability of bmDCs over 72 hrs loaded with various amounts of FMuLV. Data represents the means ± S.D. of two independent experiments.

In addition to the above experiment with FMuLV, bmDCs were also infected with fluorescent FMuLV (wFMuLV) at various MOIs. As expected, the level of infection increased with the increasing viral loads; the infection was dose-dependent and higher viral loads produced elevated levels of infection (Fig. 4.5.2). It was observed that various sub-populations were infected differently by the virus. The immature DCs were found to bear significantly higher viral infections in comparison to the mature population. Our findings are in line with the fact that immature DC phenotype bears highly efficient antigen uptake machinery which is down- regulated during the maturation process of DCs; therefore, the infection levels in immature bmDCs were always higher compared to those of mature DCs, as depicted in Fig. 4.5.2.

Results

Fig. 4.5.2: Infection pattern of sub-populations of bmDCs (immature bmDCs, iDCs; mature bmDCs, mDCs) 24 hpi loaded with various amounts of wFMuLV. Data represents the means ± S.D. of three independent experiments.

4.6. Productive infection of bmDCs As a next step we analyzed the ability of the infected bmDCs to produce the virus in the SN. Therefore, bmDCs were infected with various MOIs of FMuLV, the input virus was washed away 24 hpi and the virus containing SNs were collected five days after the infection. The SNs were loaded onto dunni cells for IC assay; the resulting levels of infection in bmDCs are shown in Fig. 4.6.1. As expected, the concentration of virus in the collected SNs increased along with the increasing viral loads from MOI of 0.001 to 0.01, but strikingly, a further increase in the viral loads resulted in a gradual reduction in the virus concentration of these SNs. At the highest MOI analyzed, i.e., 1, the concentration of virus in SN was significantly reduced. This observation of reduction in the virus concentration in SNs at higher MOIs could be due to a reduced ability of the infected bmDCs to release the virus, or it may be a consequence of a diminished capability of the released virus to further infect the dunni cells.

Results

Fig. 4.6.1: FMuLV infection of bmDCs at several MOIs. Values shown are the means ± S.D. of nine independent experiments.

4.7. RT-PCR of FMuLV-loaded bmDCs SNs In the above experiments with flow cytometry we observed that higher viral loads resulted in higher infection of bmDCs, however, it was seen in the later experiments for productive infection of bmDCs, that the concentration of virus was dramatically reduced in the SNs obtained from DCs loaded with high virus doses. This could be either due to lack of ability of DCs to release the virus or probably the virus was released but was not able to further infect the cells. To investigate the presence of virus particles in the SNs, RT-PCR was performed with the SNs obtained 5 days post infection from bmDCs infected with various MOIs of FMuLV. The findings are shown in Fig. 4.7.1. The results suggested that virus concentration was not reduced in the SNs from DCs loaded with higher virus doses compared to the SNs from DCs loaded with lower doses. This observation points towards the explanation that the concentration of virus does not reduce but the virus losses its ability to infect the cells, or in the other words, the concentration of infection-competent virus is reduced in the SNs obtained from DCs which were loaded with higher virus dose.

Results

Fig. 4.7.1: RT-PCR of SNs of bmDCs 5 dpi, infected with FMuLV at several MOIs. Results depict the relative concentration of FMuLV SNs compared to standard virus stock of known value.

4.8. Opsonization of FMuLV-Virus capture assay FMuLV was opsonized either with complement or IgG to study the effects of opsonization on the infection of bmDCs and their consequent ability to activate CTLs. Virus capture assay was performed to confirm the opsonization pattern of FMuLV, FMuLV-C and FMuLV-IgG. The amount of captured FMuLV was determined by means of RT-PCR using FV-specific primers and fluorescently labelled probes. Capture assays with complement C3-specific antibodies were able to bind FMuLV-C to much higher extent compared to FMuLV or FMuLV-IgG, revealing the presence of C3 fragments only on effectively opsonized virus, as presented in Fig. 4.8.1 A. While, in the capture assays with mouse IgG-specific antibodies, FMuLV-IgG induced a significant IgG-deposition; FMuLV and FMuLV-C did not show IgG-deposition; findings are represented in Fig. 4.8.1 B. Hence, the assay confirmed that virus stocks were successfully opsonized.

Results

(A) (B)

Fig. 4.8.1: Virus capture assay. Deposition of complement C3 (A) and IgG (B) on viral particles.

4.9. Productive infection of bmDCs with differntially opsonized FMuLV To study the effect of complement opsonization on the ability of bmDCs to get infected with the opsonized virus, the DCs were co-incubated with differentially opsonized FMuLV for five days. SNs were collected and IC assay was performed to determine the levels of infection. The results revealed a productive infection of bmDCs with complement opsonized and non- opsonized virus. However, significantly higher titers of virus were recovered from culture SNs of DCs infected with complement-opsonized FMuLV (Fig. 4.9.1). The findings indicated that complement opsonization significantly improved the virus infection of bmDCs compared to the non-opsonized virus, at least at the MOIs of 0.1 and 0.01. As observed previously in the above experiments, the infection with higher MOI reduced the concentration of infection- competent virus in the SN, and even complement opsonization could not significantly rescue this drop in the virus concentration (Fig. 4.9.2).

Results

Fig. 4.9.1: Infection of bmDCs with complement-opsonized and non-opsonized FMuLV at MOI 0.1 and 0.01. Data represents the means ± S.D. of nine independent experiments.

Additionally, we studied the capacity of IgG opsonized virus preparations to productively infect bmDCs. In contrast to the complement opsonized FMuLV, virus opsonized with FV- specific IgG further diminished the level of infection in bmDCs in comparison to non- opsonized virus, even at the MOIs at which complement opsonization of virus resulted in a significant elevation of infection level in DCs. The results are illustrated in Fig. 4.9.2.

Results

Fig. 4.9.2: Infection of bmDCs with complement- and IgG-opsonized FMuLV at various MOIs. Non-opsonized virus was used as control. Data represents the means ± S.D. of nine independent experiments.

4.10. Characterization of CD11b-/- and CD11c-/- mice derived DCs To confirm the successful knock-out of CD11b and CD11c, RT-PCR and FACS analysis were performed. The results proved the absence of CD11b and CD11c expression on the DCs (Fig. 4.10.1 A and B). As CR3 and CR4 are closely related entities and share the β-chain, CD18, of the receptor, therefore a possibility exist that in the absence of one α-chain, CD11b or CD11c, the expression of the second α-chain might increase in order to compensate for the deletion of one of its counterparts. To verify this hypothesis, we also looked for the expression of CD11c (Fig. 4.10.1 A) on CD11b-/- DCs and of CD11b (Fig. 4.10.1 B) on CD11c-/- DCs. To further confirm the depletion of CD11b or CD11c, their expression was also tested in the presence of DC maturation stimulators, LPS or CpG. The obtained results confirmed the successful depletion of CD11b or CD11c in immature as well as mature bmDCs.

Results

(A) (B) 50000 15000 wt wt CD11b ko CD11b ko CD11c ko CD11c ko 40000

10000

c b

1 30000

I F

20000 F

M M 5000

10000

0 0 C C C C C C C C C C C C C C C C C C D -D -D D -D -D D -D -D D -D -D D -D -D D -D -D S G S G S G S G S G S G P p P p P p P p P p P p L C L C L C L C L C L C

Fig. 4.10.1: Expression of CD11c (A) and CD11b (B) on day 8 bmDCs derived from wt, CD11b-/- and CD11c-/- mice in the presence and absence of DC-maturation stimulator molecules, LPS (10 µg/mL) or CpG (4 µg/mL).

4.11. Maturation behavior of CD11b-/- and CD11c-/- mice derived DCs In order to confirm that depletion of CD11b or CD11c molecules does not affect the maturation behavior of bmDCs, the expression of typical maturation and co-stimulation markers, MHC II and CD86, was analyzed in DCs derived from wt, CD11b and CD11c knock-out (KO) mice. The results obtained are presented in the Fig. 4.11.1. The expression of the maturation markers in DCs derived from knock-out mice was similar to those derived from wt mice. This suggests that the absence of the α-chain of either CR3 or CR4 has no significant effect on the maturation pattern of bmDCs; they undergo the process of maturation in a normal way and express antigen presenting and co-stimulatory molecules at a similar level to that of wt mouse-derived DCs.

Results

80 wt CD11b ko CD11c ko

H M

+ 40

C %

0 C C C C C C C C C D -D -D D -D -D D -D -D S G S G S G P p P p P p L C L C L C

Fig. 4.11.1: Expression of maturation markers on day 8 bmDCs derived from wt, CD11b-/- and CD11c-/- mice in the presence and absence of DC-maturation stimulator molecules, LPS (10 µg/mL) or CpG (4 µg/mL).

4.12. CD8+ T cell activation by CD11b-/- and CD11c-/- mice derived DCs Day 8 bmDCs were infected with opsonized or non-opsonized FMuLV which were co- cultured on day 9 with CD8+ T cells derived from FV-specific TCRtg mice. The next day these cells were collected and analyzed for the expression of T cell activation markers, CD25 and CD69 (Fig. 4.12.1). The experimental outcomes revealed that both complement opsonized and non-opsonized FMuLV loaded-DCs derived from CD11c-/- mice were able to activate the CD8+ T cells in a fashion comparable to those derived from wt mice. However, virus loaded DCs derived from CD11b-/- mice could activate a significantly lower number of CD8+ T cells compared to those derived from wt mice. This indicates the importance of CD11b in the process of T cell activation in comparison to its counterpart, CD11c. To further confirm these findings, we normalized the values obtained by non-opsonized FMuLV-loaded DCs at 100. Still, the absence of CD11b exhibited low capability to activate the CD8+ T cells (Fig. 4.12.1 B).

Results

(A) 25 wt CD11b ko CD11c ko

6 D

C 15

C %

D C 5

0 C C C C C C C C C D D D D D D D D D m m m m m m m m m b /b /b b /b /b b /b /b V V V V V V L L L L L L u u u u u u M M M M M M F -F F -F F -F (B) C C C

300 wt

) CD11b ko

d e

z CD11c ko

m r

o 200

(

5 2

D 100

D C

0 C C C C C C C C C D D D D D D D D D m m m m m m m m m b /b /b b /b /b b /b /b V V V V V V L L L L L L u u u u u u M M M M M M F -F F -F F -F C C C

Fig. 4.12.1: Activation of CD8+ T cells by opsonized or non-opsonized FMuLV-loaded bmDCs derived from wt, CD11b-/- and CD11c-/- mice (A). The activation induced by non- opsonized FMuLV-loaded DCs was normalized to 100 (B).

Discussion

5. DISCUSSION

The present study demonstrates that not only the different patterns of FV opsonization have a profound influence on the infection of DCs but also the initial viral load has an impact on the DC infection by FV. Furthermore, in this study we describe the crucial role of CR3 (CD11b) in the induction of efficient retroviral-specific CTL responses as a result of FV infection of bmDCs.

The involvement of complement in the induction of CD8+ T cell responses against viral infections has been demonstrated in some previous studies (Kopf et al., 2002; Mehlhop and Diamond, 2006; Suresh et al., 2003). However, the mechanism involved with a major focus on complement receptors, in the generation of the CTL responses has not been fully described. Earlier, in a study from our group it was illustrated that opsonization of virus particles with complement proteins expands the infection of DCs not only in vitro but in vivo as well (Banki et al., 2010). Complement plays a role in facilitating presentation of antigens by DCs for the activation and differentiation of virus-specific CTLs. In another study it was shown that HIV-specific CD8+ T cells generated by repeated prime-boosting with complement opsonized HIV-loaded DCs possessed the ability to secrete IFN- and exhibited antiviral activity against HIV-infected CD4+ T cells (Betts et al., 2004). The DCs exposed to non-opsonized HIV were able to prime the T cells to a much lower extent. The particular ability of DCs to prime naïve CTL as response to both exogenous and endogenous antigens has been described in the literature (Jung et al., 2002; Probst and van den Broek, 2005). Macrophages or B cells infected with Lymphocytic choriomeningitis virus (LCMV) did not elicit CD8+ T cell response, on the other hand, LCMV infected DCs were able to launch a strong CTL response (Probst and van den Broek, 2005). In a study with FV, it was demonstrated that the induction of virus-specific CD8+ T cells was significantly diminished as a result of depletion of DCs in FV-infected mice (Browne and Littman, 2009). The complement-opsonized virus interacts with CRs present on APCs. It has been shown that iC3b fragments on the surface of virus influence and promote the infection of monocytes, macrophages, B cells and DCs which express CR3 and/or CR4 (Bajtay et al., 2004; Bila et al., 2011; Pruenster et al., 2005; Stoiber et al., 1997; Thieblemont et al., 1995; Wilflingseder et al., 2005).

The presentation of viral antigens synthesized endogenously through MHC class I pathway might be linked to the infection of DCs in order to mount a response by naïve CD8+ T cells

Discussion

(Banki et al., 2010). Loading DCs with opsonized HIV resulted in an improvement of their infection in comparison to the non-opsonized HIV (Wilflingseder et al., 2007). Therefore, DCs infected with opsonized HIV can process the antigens endogenously via MHC class I pathway and are thus able to prime naïve CD8+ T cells in order to induce an efficient CTL response. C3-/- mice when challenged with FV demonstrated low infection of DCs followed by poor mounting of virus-specific CTL response which resulted in significantly higher acute viremia in comparison to the wt mice (Banki et al., 2010). Moreover, it was described in another study that presence of antibodies against CR3 resulted in a reduction of HIV infection in vitro (Stoiber et al., 1997; Stoiber et al., 2008b). It was demonstrated that opsonized HIV showed a significantly enhanced internalization of virus by DCs in comparison to non- opsonized HIV, although the amount of both opsonized and non-opsonized HIV was similar (Tjomsland et al., 2011). The difference in the uptake of virus might be due to the differential usage of receptors within the integrin and C-type receptor family. The higher uptake of complement opsonized virus is correlated to the improved productive infection of the DCs. This better infectivity is possibly due to decreased proteolysis and prolonged infection time which could be because of different routing of the virus in the cells as a result of using different receptors by opsonized- and non-opsonized viruses. An additional explanation could be the higher affinity of complement opsonized virus to the lipid-rich domains of cell membrane which might facilitate the binding of the virus to the receptors (Stoiber et al., 2008a).

The improved internalization of complement opsonized virus is probably through CR3 or CR4. It is not easy to investigate the relative contribution of CR3 (CD11b) and CR4 (CD11c) in this mechanism, for reasons like, co-expression of these receptors and the non-availability of appropriate blocking antibodies (Stoiber et al., 2008b). Therefore, to selectively investigate the role of CR3 and CR4 on DCs for antigen presentation and the induction of CTL responses, a knock out mouse model was used. In our study, we observed the crucial role of CR3 (CD11b) which it plays in the induction of FV-specific CD8+ T cells. It was shown that depletion of CD11c on DCs had no significant difference on the activation of FV-specific CD8+ T cells in comparison to the wt DCs. On the other hand, when CD11b was depleted from the DCs and these DCs after loading with FMuLV-C were then co-cultured with CD8+ T cells, there was a significant reduction in the activation of FV-specific CD8+ T cells compared to the DCs derived from wt mouse.

Discussion

Our findings are in line with a previous study where it was demonstrated that complement opsonized HIV significantly amplified the productive infection of human monocyte-derived DCs, but this enhancement in the infection was completely abolished in the presence of a monoclonal antibody specific for the ligand-bonding site of CD11b (Bajtay et al., 2004). The role of CR3 has been studied where the inhibition of CD11b by monoclonal antibodies resulted in the loss of infection with HIV in monocytes (Bouhlal et al., 2001; Kacani et al., 1998) and in peripheral blood mononuclear cells (PBMCs) (Stoiber et al., 1997). There is a possibility that binding of FV coated with complement to CD11b of CR3 may trigger a cascade of signals and directs the virus to compartments in DCs which are favorable for the productive infection of the cells; which then leads to more efficient induction of virus-specific CTL responses.

In our study we also observed that in contrast to the enhanced infection of DCs as a result of complement opsonization of the virus, the opsonization of FMuLV with IgG lead to an abrogated infection of DCs. Our results correlate with the findings of a study in which an impaired provirus formation and infection of human DCs was demonstrated as a result of coating HIV with IgG. The IgG opsonization also interfered with the long term transfer of HIV from DCs to T cells (Wilflingseder et al., 2007). This inhibition of infection was not due to the neutralizing antibodies.

It was demonstrated in our findings that higher viral loads resulted in a strikingly diminished infectious viremia during productive infection of DCs, and this decrease in the virus concentration could not be rescued even by the complement opsonization of the virus. The findings from the bmDCs viability experiments ruled out the possibility of cell death as a reason for low infection levels at the higher viral loads. The FACS data showed that DCs were infected at higher viral doses; this suggests that the infected DCs were either not able to release virus or the released virus somehow lost its infectivity. The findings from RT-PCR indicated that the virus concentration was not reduced comparatively at higher MOIs, suggesting the virus was not infection competent anymore. One possible reason for the infectivity reduction could be some kind of host restriction factor which might be involved in the abrogation of the ability of FMuLV to infect the cells. There have been several host- encoded restriction factors identified that work as effective inhibitors of retroviral replication. Some of the inhibitory factors for retroviral infection include, APOBEC3, SAMHD1, TRIM5, tetherin, MOV10, Fv1 and cellular microRNAs (Harris et al., 2012; Malim and Bieniasz, 2012; Rein, 2011; Simon et al., 2015; Wang et al., 2016; Zheng et al., 2012).

Discussion

DCs express several intrinsic defense machineries to inhibit HIV-1 replication; therefore the virus is able to productively infect DCs at very low levels. Recently, it was demonstrated that complement-opsonized HIV-1 efficiently bypassed the restriction by SAMHD1 and productively infected DCs. The efficient infection of DCs by opsonized virus correlated with the elevated phosphorylation of SAMHD1 (Posch et al., 2015). In another study it was illustrated that mouse SAMHD1, similar to human SAMHD1, depletes the deoxy-nucleotide triphosphate (dNTP) pool below the levels required for retroviral reverse transcription, in murine cells and thus restricts retroviral infection (Behrendt et al., 2013). SAMHD1 dependent retroviral restriction in mice is regulated through its phosphorylation (Wittmann et al., 2015). Moreover, in a study mouse tetherin was demonstrated to inhibit MLV release from infected cells in vitro by retaining the virus on the cell membrane (Goffinet et al., 2010; Li et al., 2016; Rein, 2011).

Our results fit more to the restriction mechanism of APOBEC3 which renders the released particles as noninfectious (Cullen, 2006; Halemano et al., 2013). APOBEC3 proteins are incorporated into retrovirus particles and when this APOBEC3-carrying virus infects a new host cell, these proteins interfere with viral replication during reverse transcription (Low et al., 2009; Rein, 2011). APOBEC3 activity does not decrease the virus release, whereas the infectivity of the output virus is significantly inhibited (Smith et al., 2011). In a study it was illustrated that majority of FV circulating in wt C57/BL6 plasma were noninfectious. The infectious virus titer was 30-fold higher in APOBEC3 KO mice in contrast to wt mice (Harper et al., 2013). Still another study demonstrated that APOBEC3 deficient C57/BL6 mice possessed 100-fold higher numbers of FMuLV-producing cells in bone marrow and spleen, compared to wt mice (Takeda et al., 2008). It seems that more than one restriction factors work in concert to efficiently inhibit the infection of bmDCs in one way or another.

In summary, our data imply that depending upon the opsonization pattern, FV, a model retrovirus; exert diverse modulatory effects on DCs. The virus activates DCs in a complement-dependent manner in order to induce cellular immune responses. The infectivity of the output virus from infected DCs is also depending on the initial viral loads; higher viral loads lead to diminished infection-competent virus concentration released from DCs. Moreover, CR3 plays a crucial role in the infection of APCs and in the induction of cellular immune responses. Our results may provide valuable information about the approaches to target antigens to DCs in order to enhance vaccine efficacy or to develop novel and improved vaccine candidates.

References

6. REFERENCES

Arvieux, J., Yssel, H., Colomb, M.G., 1988. Antigen-bound C3b and C4b enhance antigen- presenting cell function in activation of human T-cell clones. Immunology 65, 229-235.

Bajtay, Z., Speth, C., Erdei, A., Dierich, M.P., 2004. Cutting edge: productive HIV-1 infection of dendritic cells via complement receptor type 3 (CR3, CD11b/CD18). J Immunol 173, 4775-4778.

Banchereau, J., Briere, F., Caux, C., Davoust, J., Lebecque, S., Liu, Y.J., Pulendran, B., Palucka, K., 2000. Immunobiology of dendritic cells. Annu Rev Immunol 18, 767-811.

Banchereau, J., Steinman, R.M., 1998. Dendritic cells and the control of immunity. Nature 392, 245-252.

Banki, Z., Posch, W., Ejaz, A., Oberhauser, V., Willey, S., Gassner, C., Stoiber, H., Dittmer, U., Dierich, M.P., Hasenkrug, K.J., Wilflingseder, D., 2010. Complement as an endogenous adjuvant for dendritic cell-mediated induction of retrovirus-specific CTLs. PLoS Pathog 6, e1000891.

Behrendt, R., Schumann, T., Gerbaulet, A., Nguyen, L.A., Schubert, N., Alexopoulou, D., Berka, U., Lienenklaus, S., Peschke, K., Gibbert, K., Wittmann, S., Lindemann, D., Weiss, S., Dahl, A., Naumann, R., Dittmer, U., Kim, B., Mueller, W., Gramberg, T., Roers, A., 2013. Mouse SAMHD1 has antiretroviral activity and suppresses a spontaneous cell-intrinsic antiviral response. Cell Rep 4, 689-696.

Betts, M.R., Price, D.A., Brenchley, J.M., Lore, K., Guenaga, F.J., Smed-Sorensen, A., Ambrozak, D.R., Migueles, S.A., Connors, M., Roederer, M., Douek, D.C., Koup, R.A., 2004. The functional profile of primary human antiviral CD8+ T cell effector activity is dictated by cognate peptide concentration. J Immunol 172, 6407-6417.

Bevan, M.J., 1976. Cross-priming for a secondary cytotoxic response to minor H antigens with H-2 congenic cells which do not cross-react in the cytotoxic assay. J Exp Med 143, 1283-1288.

Bexborn, F., Andersson, P.O., Chen, H., Nilsson, B., Ekdahl, K.N., 2008. The tick-over theory revisited: formation and regulation of the soluble alternative complement C3 convertase (C3(H2O)Bb). Mol Immunol 45, 2370-2379.

References

Bila, C., Oberhauser, V., Ammann, C.G., Ejaz, A., Huber, G., Schimmer, S., Messer, R., Pekna, M., von Laer, D., Dittmer, U., Hasenkrug, K.J., Stoiber, H., Banki, Z., 2011. Complement opsonization enhances friend virus infection of B cells and thereby amplifies the virus-specific CD8+ T cell response. J Virol 85, 1151-1155.

Bilsland, C.A., Diamond, M.S., Springer, T.A., 1994. The leukocyte integrin p150,95 (CD11c/CD18) as a receptor for iC3b. Activation by a heterologous beta subunit and localization of a ligand recognition site to the I domain. J Immunol 152, 4582-4589.

Blackmore, T.K., Hellwage, J., Sadlon, T.A., Higgs, N., Zipfel, P.F., Ward, H.M., Gordon, D.L., 1998. Identification of the second heparin-binding domain in human complement factor H. J Immunol 160, 3342-3348.

Bondurant, M.C., Koury, M.J., Krantz, S.B., 1985. The Fv-2 gene controls induction of erythroid burst formation by Friend virus infection in vitro: studies of growth regulators and viral replication. J Gen Virol 66 ( Pt 1), 83-96.

Bouhlal, H., Galon, J., Kazatchkine, M.D., Fridman, W.H., Sautes-Fridman, C., Haeffner Cavaillon, N., 2001. Soluble CD16 inhibits CR3 (CD11b/CD18)-mediated infection of monocytes/macrophages by opsonized primary R5 HIV-1. J Immunol 166, 3377-3383.

Browne, E.P., Littman, D.R., 2009. Myd88 is required for an antibody response to retroviral infection. PLoS Pathog 5, e1000298.

Bubbers, J.E., Lilly, F., 1977. Selective incorporation of H-2 antigenic determinants into Friend virus particles. Nature 266, 458-459.

Caminschi, I., Ahmet, F., Heger, K., Brady, J., Nutt, S.L., Vremec, D., Pietersz, S., Lahoud, M.H., Schofield, L., Hansen, D.S., O'Keeffe, M., Smyth, M.J., Bedoui, S., Davey, G.M., Villadangos, J.A., Heath, W.R., Shortman, K., 2007. Putative IKDCs are functionally and developmentally similar to natural killer cells, but not to dendritic cells. J Exp Med 204, 2579-2590.

Carroll, M.C., 2004. The complement system in regulation of adaptive immunity. Nat Immunol 5, 981-986.

Cavanagh, L.L., Von Andrian, U.H., 2002. Travellers in many guises: the origins and destinations of dendritic cells. Immunol Cell Biol 80, 448-462.

References

Chen, W., Qin, H., Chesebro, B., Cheever, M.A., 1996. Identification of a gag-encoded cytotoxic T-lymphocyte epitope from FBL-3 leukemia shared by Friend, Moloney, and Rauscher murine leukemia virus-induced tumors. J Virol 70, 7773-7782.

Chesebro, B., Miyazawa, M., Britt, W.J., 1990. Host genetic control of spontaneous and induced immunity to Friend murine retrovirus infection. Annu Rev Immunol 8, 477-499.

Chmouryguina, I., Suvorov, A., Ferrieri, P., Cleary, P.P., 1996. Conservation of the C5a peptidase genes in group A and B streptococci. Infect Immun 64, 2387-2390.

Choi, J., Nham, S.U., 2002. Loops within the CD11c I domain critical for specific recognition of fibrinogen. Biochem Biophys Res Commun 292, 756-760.

Chung, S.W., Wolff, L., Ruscetti, S.K., 1989. Transmembrane domain of the envelope gene of a polycythemia-inducing retrovirus determines erythropoietin-independent growth. Proc Natl Acad Sci U S A 86, 7957-7960.

Cmarik, J., Ruscetti, S., 2010. Friend Spleen Focus-Forming Virus Activates the Tyrosine Kinase sf-Stk and the Transcription Factor PU.1 to Cause a Multi-Stage Erythroleukemia in Mice. Viruses 2, 2235-2257.

Constantinescu, S.N., Liu, X., Beyer, W., Fallon, A., Shekar, S., Henis, Y.I., Smith, S.O., Lodish, H.F., 1999. Activation of the erythropoietin receptor by the gp55-P viral envelope protein is determined by a single amino acid in its transmembrane domain. EMBO J 18, 3334- 3347.

Corbin, A., Prats, A.C., Darlix, J.L., Sitbon, M., 1994. A nonstructural gag-encoded glycoprotein precursor is necessary for efficient spreading and pathogenesis of murine leukemia viruses. J Virol 68, 3857-3867.

Cullen, B.R., 2006. Role and mechanism of action of the APOBEC3 family of antiretroviral resistance factors. J Virol 80, 1067-1076.

Denton, P.W., Garcia, J.V., 2009. Novel humanized murine models for HIV research. Curr HIV/AIDS Rep 6, 13-19.

Diamond, M.S., Garcia-Aguilar, J., Bickford, J.K., Corbi, A.L., Springer, T.A., 1993. The I domain is a major recognition site on the leukocyte integrin Mac-1 (CD11b/CD18) for four distinct adhesion ligands. J Cell Biol 120, 1031-1043.

References

Dittmer, U., Hasenkrug, K.J., 2001. Cellular and molecular mechanisms of vaccine-induced protection against retroviral infections. Curr Mol Med 1, 431-436.

Dittmer, U., He, H., Messer, R.J., Schimmer, S., Olbrich, A.R., Ohlen, C., Greenberg, P.D., Stromnes, I.M., Iwashiro, M., Sakaguchi, S., Evans, L.H., Peterson, K.E., Yang, G., Hasenkrug, K.J., 2004. Functional impairment of CD8(+) T cells by regulatory T cells during persistent retroviral infection. Immunity 20, 293-303.

Dubois, B., Vanbervliet, B., Fayette, J., Massacrier, C., Van Kooten, C., Briere, F., Banchereau, J., Caux, C., 1997. Dendritic cells enhance growth and differentiation of CD40- activated B lymphocytes. J Exp Med 185, 941-951.

Dunkelberger, J.R., Song, W.C., 2010. Complement and its role in innate and adaptive immune responses. Cell Res 20, 34-50.

Ehlers, M.R., 2000. CR3: a general purpose adhesion-recognition receptor essential for innate immunity. Microbes Infect 2, 289-294.

Ejaz, A., Ammann, C.G., Werner, R., Huber, G., Oberhauser, V., Horl, S., Schimmer, S., Dittmer, U., von Laer, D., Stoiber, H., Banki, Z., 2012. Targeting viral antigens to CD11c on dendritic cells induces retrovirus-specific T cell responses. PLoS One 7, e45102.

Erdei, A., Isaak, A., Torok, K., Sandor, N., Kremlitzka, M., Prechl, J., Bajtay, Z., 2009. Expression and role of CR1 and CR2 on B and T lymphocytes under physiological and autoimmune conditions. Mol Immunol 46, 2767-2773.

Favoreel, H.W., Van de Walle, G.R., Nauwynck, H.J., Pensaert, M.B., 2003. Virus complement evasion strategies. J Gen Virol 84, 1-15.

Fingeroth, J.D., Heath, M.E., Ambrosino, D.M., 1989. Proliferation of resting B cells is modulated by CR2 and CR1. Immunol Lett 21, 291-301.

Frank, M.M., 2001. Annihilating host defense. Nat Med 7, 1285-1286.

Freed, E.O., Risser, R., 1987. The role of envelope glycoprotein processing in murine leukemia virus infection. J Virol 61, 2852-2856.

Freeley, S., Kemper, C., Le Friec, G., 2016. The "ins and outs" of complement-driven immune responses. Immunol Rev 274, 16-32.

References

Friend, C., 1957. Cell-free transmission in adult Swiss mice of a disease having the character of a leukemia. J Exp Med 105, 307-318.

Fujisawa, R., McAtee, F.J., Favara, C., Hayes, S.F., Portis, J.L., 2001. N-terminal cleavage fragment of glycosylated Gag is incorporated into murine oncornavirus particles. J Virol 75, 11239-11243.

Goff, S.P., 2007. Host factors exploited by retroviruses. Nat Rev Microbiol 5, 253-263.

Goffinet, C., Schmidt, S., Kern, C., Oberbremer, L., Keppler, O.T., 2010. Endogenous CD317/Tetherin limits replication of HIV-1 and murine leukemia virus in rodent cells and is resistant to antagonists from primate viruses. J Virol 84, 11374-11384.

Guan, R., Aiyer, S., Cote, M.L., Xiao, R., Jiang, M., Acton, T.B., Roth, M.J., Montelione, G.T., 2017. X-ray crystal structure of the N-terminal region of Moloney murine leukemia virus integrase and its implications for viral DNA recognition. Proteins.

Guo, R.F., Ward, P.A., 2005. Role of C5a in inflammatory responses. Annu Rev Immunol 23, 821-852.

Halemano, K., Harper, M.S., Guo, K., Li, S.X., Heilman, K.J., Barrett, B.S., Santiago, M.L., 2013. Humoral immunity in the Friend retrovirus infection model. Immunol Res 55, 249-260.

Harper, M.S., Barrett, B.S., Smith, D.S., Li, S.X., Gibbert, K., Dittmer, U., Hasenkrug, K.J., Santiago, M.L., 2013. IFN-alpha treatment inhibits acute Friend retrovirus replication primarily through the antiviral effector molecule Apobec3. J Immunol 190, 1583-1590.

Harris, R.S., Hultquist, J.F., Evans, D.T., 2012. The restriction factors of human immunodeficiency virus. J Biol Chem 287, 40875-40883.

Hasenkrug, K.J., 1999. Lymphocyte deficiencies increase susceptibility to friend virus- induced erythroleukemia in Fv-2 genetically resistant mice. J Virol 73, 6468-6473.

Hasenkrug, K.J., Brooks, D.M., Chesebro, B., 1995. Passive immunotherapy for retroviral disease: influence of major histocompatibility complex type and T-cell responsiveness. Proc Natl Acad Sci U S A 92, 10492-10495.

Hasenkrug, K.J., Brooks, D.M., Dittmer, U., 1998a. Critical role for CD4(+) T cells in controlling retrovirus replication and spread in persistently infected mice. J Virol 72, 6559- 6564. 71

References

Hasenkrug, K.J., Brooks, D.M., Robertson, M.N., Srinivas, R.V., Chesebro, B., 1998b. Immunoprotective determinants in friend murine leukemia virus envelope protein. Virology 248, 66-73.

Hasenkrug, K.J., Chesebro, B., 1997. Immunity to retroviral infection: the Friend virus model. Proc Natl Acad Sci U S A 94, 7811-7816.

Hasenkrug, K.J., Dittmer, U., 2000. The role of CD4 and CD8 T cells in recovery and protection from retroviral infection: lessons from the Friend virus model. Virology 272, 244- 249.

Hasenkrug, K.J., Dittmer, U., 2007. Immune control and prevention of chronic Friend retrovirus infection. Front Biosci 12, 1544-1551.

Heath, W.R., Belz, G.T., Behrens, G.M., Smith, C.M., Forehan, S.P., Parish, I.A., Davey, G.M., Wilson, N.S., Carbone, F.R., Villadangos, J.A., 2004. Cross-presentation, dendritic cell subsets, and the generation of immunity to cellular antigens. Immunol Rev 199, 9-26.

Heath, W.R., Carbone, F.R., 2001. Cross-presentation in viral immunity and self-tolerance. Nat Rev Immunol 1, 126-134.

Heinen, S., Hartmann, A., Lauer, N., Wiehl, U., Dahse, H.M., Schirmer, S., Gropp, K., Enghardt, T., Wallich, R., Halbich, S., Mihlan, M., Schlotzer-Schrehardt, U., Zipfel, P.F., Skerka, C., 2009. Factor H-related protein 1 (CFHR-1) inhibits complement C5 convertase activity and terminal complex formation. Blood 114, 2439-2447.

Helft, J., Bottcher, J., Chakravarty, P., Zelenay, S., Huotari, J., Schraml, B.U., Goubau, D., Reis e Sousa, C., 2015. GM-CSF Mouse Bone Marrow Cultures Comprise a Heterogeneous Population of CD11c(+)MHCII(+) Macrophages and Dendritic Cells. Immunity 42, 1197- 1211.

Hubo, M., Trinschek, B., Kryczanowsky, F., Tuettenberg, A., Steinbrink, K., Jonuleit, H., 2013. Costimulatory molecules on immunogenic versus tolerogenic human dendritic cells. Front Immunol 4, 82.

Inaba, K., Inaba, M., Romani, N., Aya, H., Deguchi, M., Ikehara, S., Muramatsu, S., Steinman, R.M., 1992. Generation of large numbers of dendritic cells from mouse bone marrow cultures supplemented with granulocyte/macrophage colony-stimulating factor. J Exp Med 176, 1693-1702. 72

References

Inal, J.M., 2004. Parasite interaction with host complement: beyond attack regulation. Trends Parasitol 20, 407-412.

Iwama, A., Okano, K., Sudo, T., Matsuda, Y., Suda, T., 1994. Molecular cloning of a novel receptor tyrosine kinase gene, STK, derived from enriched hematopoietic stem cells. Blood 83, 3160-3169.

Iwashiro, M., Peterson, K., Messer, R.J., Stromnes, I.M., Hasenkrug, K.J., 2001. CD4(+) T cells and gamma interferon in the long-term control of persistent friend retrovirus infection. J Virol 75, 52-60.

Joffre, O.P., Segura, E., Savina, A., Amigorena, S., 2012. Cross-presentation by dendritic cells. Nat Rev Immunol 12, 557-569.

Joiner, K., Brown, E., Hammer, C., Warren, K., Frank, M., 1983. Studies on the mechanism of bacterial resistance to complement-mediated killing. III. C5b-9 deposits stably on rough and type 7 S. pneumoniae without causing bacterial killing. J Immunol 130, 845-849.

Jung, S., Unutmaz, D., Wong, P., Sano, G., De los Santos, K., Sparwasser, T., Wu, S., Vuthoori, S., Ko, K., Zavala, F., Pamer, E.G., Littman, D.R., Lang, R.A., 2002. In vivo depletion of CD11c+ dendritic cells abrogates priming of CD8+ T cells by exogenous cell- associated antigens. Immunity 17, 211-220.

Jung, Y.T., Wu, T., Kozak, C.A., 2004. Novel host range and cytopathic variant of ecotropic Friend murine leukemia virus. J Virol 78, 12189-12197.

Kacani, L., Frank, I., Spruth, M., Schwendinger, M.G., Mullauer, B., Sprinzl, G.M., Steindl, F., Dierich, M.P., 1998. Dendritic cells transmit human immunodeficiency virus type 1 to monocytes and monocyte-derived macrophages. J Virol 72, 6671-6677.

Keizer, G.D., Te Velde, A.A., Schwarting, R., Figdor, C.G., De Vries, J.E., 1987. Role of p150,95 in adhesion, migration, chemotaxis and phagocytosis of human monocytes. Eur J Immunol 17, 1317-1322.

Khera, R., Das, N., 2009. Complement Receptor 1: disease associations and therapeutic implications. Mol Immunol 46, 761-772.

Klos, A., Tenner, A.J., Johswich, K.O., Ager, R.R., Reis, E.S., Kohl, J., 2009. The role of the anaphylatoxins in health and disease. Mol Immunol 46, 2753-2766.

References

Kolev, M., Le Friec, G., Kemper, C., 2014. Complement--tapping into new sites and effector systems. Nat Rev Immunol 14, 811-820.

Kopf, M., Abel, B., Gallimore, A., Carroll, M., Bachmann, M.F., 2002. Complement component C3 promotes T-cell priming and lung migration to control acute influenza virus infection. Nat Med 8, 373-378.

Kumar, V., Bennett, M., Eckner, R.J., 1974. Mechanisms of genetic resistance to friend virus leukemia in mice. J Exp Med 139, 1093-1109.

Kwan, W.H., van der Touw, W., Paz-Artal, E., Li, M.O., Heeger, P.S., 2013. Signaling through C5a receptor and C3a receptor diminishes function of murine natural regulatory T cells. J Exp Med 210, 257-268.

Lambris, J.D., Ricklin, D., Geisbrecht, B.V., 2008. Complement evasion by human pathogens. Nat Rev Microbiol 6, 132-142.

Lander, M.R., Chattopadhyay, S.K., 1984. A Mus dunni cell line that lacks sequences closely related to endogenous murine leukemia viruses and can be infected by ectropic, amphotropic, xenotropic, and mink cell focus-forming viruses. J Virol 52, 695-698.

Langley, R., Wines, B., Willoughby, N., Basu, I., Proft, T., Fraser, J.D., 2005. The staphylococcal superantigen-like protein 7 binds IgA and complement C5 and inhibits IgA-Fc alpha RI binding and serum killing of bacteria. J Immunol 174, 2926-2933.

Lanier, L.L., Arnaout, M.A., Schwarting, R., Warner, N.L., Ross, G.D., 1985. p150/95, Third member of the LFA-1/CR3 polypeptide family identified by anti-Leu M5 monoclonal antibody. Eur J Immunol 15, 713-718.

Lanzavecchia, A., Sallusto, F., 2001. Regulation of T cell immunity by dendritic cells. Cell 106, 263-266.

Leslie, R.G.Q., 2001. Complement Receptors, eLS. John Wiley & Sons Ltd, Chichester.

Leslie, R.G.Q., Hansen, S., 2009. Complement Receptors, eLS. John Wiley & Sons Ltd, Chichester.

Li, J.P., Hu, H.O., Niu, Q.T., Fang, C., 1995. Cell surface activation of the erythropoietin receptor by Friend spleen focus-forming virus gp55. J Virol 69, 1714-1719.

References

Li, S., Gowans, E.J., Chougnet, C., Plebanski, M., Dittmer, U., 2008. Natural regulatory T cells and persistent viral infection. J Virol 82, 21-30.

Li, S.X., Barrett, B.S., Guo, K., Kassiotis, G., Hasenkrug, K.J., Dittmer, U., Gibbert, K., Santiago, M.L., 2016. Tetherin/BST-2 promotes dendritic cell activation and function during acute retrovirus infection. Sci Rep 6, 20425.

Liszewski, M.K., Farries, T.C., Lublin, D.M., Rooney, I.A., Atkinson, J.P., 1996. Control of the complement system. Adv Immunol 61, 201-283.

Littwitz-Salomon, E., Schimmer, S., Dittmer, U., 2017. Natural killer T cells contribute to the control of acute retroviral infection. Retrovirology 14, 5.

Low, A., Datta, S., Kuznetsov, Y., Jahid, S., Kothari, N., McPherson, A., Fan, H., 2007. Mutation in the glycosylated gag protein of murine leukemia virus results in reduced in vivo infectivity and a novel defect in viral budding or release. J Virol 81, 3685-3692.

Low, A., Okeoma, C.M., Lovsin, N., de las Heras, M., Taylor, T.H., Peterlin, B.M., Ross, S.R., Fan, H., 2009. Enhanced replication and pathogenesis of Moloney murine leukemia virus in mice defective in the murine APOBEC3 gene. Virology 385, 455-463.

Lubinski, J., Nagashunmugam, T., Friedman, H.M., 1998. Viral interference with antibody and complement. Semin Cell Dev Biol 9, 329-337.

Machinaga, A., Ishihara, S., Shirai, A., Takase-Yoden, S., 2016. Splicing of Friend Murine Leukemia Virus env-mRNA Enhances Its Ability to Form Polysomes. Front Microbiol 7, 160.

Malim, M.H., Bieniasz, P.D., 2012. HIV Restriction Factors and Mechanisms of Evasion. Cold Spring Harb Perspect Med 2, a006940.

Markiewski, M.M., Lambris, J.D., 2007. The role of complement in inflammatory diseases from behind the scenes into the spotlight. Am J Pathol 171, 715-727.

Mathern, D.R., Heeger, P.S., 2015. Molecules Great and Small: The Complement System. Clin J Am Soc Nephrol 10, 1636-1650.

Mehlhop, E., Diamond, M.S., 2006. Protective immune responses against West Nile virus are primed by distinct complement activation pathways. J Exp Med 203, 1371-1381.

References

Meri, S., Morgan, B.P., Davies, A., Daniels, R.H., Olavesen, M.G., Waldmann, H., Lachmann, P.J., 1990. Human protectin (CD59), an 18,000-20,000 MW complement lysis restricting factor, inhibits C5b-8 catalysed insertion of C9 into lipid bilayers. Immunology 71, 1-9.

Meri, T., Blom, A.M., Hartmann, A., Lenk, D., Meri, S., Zipfel, P.F., 2004. The hyphal and yeast forms of Candida albicans bind the complement regulator C4b-binding protein. Infect Immun 72, 6633-6641.

Meri, T., Hartmann, A., Lenk, D., Eck, R., Wurzner, R., Hellwage, J., Meri, S., Zipfel, P.F., 2002. The yeast Candida albicans binds complement regulators factor H and FHL-1. Infect Immun 70, 5185-5192.

Merle, N.S., Church, S.E., Fremeaux-Bacchi, V., Roumenina, L.T., 2015a. Complement System Part I - Molecular Mechanisms of Activation and Regulation. Front Immunol 6, 262.

Merle, N.S., Noe, R., Halbwachs-Mecarelli, L., Fremeaux-Bacchi, V., Roumenina, L.T., 2015b. Complement System Part II: Role in Immunity. Front Immunol 6, 257.

Moore, M.D., Cooper, N.R., Tack, B.F., Nemerow, G.R., 1987. Molecular cloning of the cDNA encoding the Epstein-Barr virus/C3d receptor (complement receptor type 2) of human B lymphocytes. Proc Natl Acad Sci U S A 84, 9194-9198.

Moreau-Gachelin, F., 2008. Multi-stage Friend murine erythroleukemia: molecular insights into oncogenic cooperation. Retrovirology 5, 99.

Mueller-Ortiz, S.L., Wang, D., Morales, J.E., Li, L., Chang, J.Y., Wetsel, R.A., 2009. Targeted disruption of the gene encoding the murine small subunit of carboxypeptidase N (CPN1) causes susceptibility to C5a anaphylatoxin-mediated shock. J Immunol 182, 6533- 6539.

Myones, B.L., Dalzell, J.G., Hogg, N., Ross, G.D., 1988. Neutrophil and monocyte cell surface p150,95 has iC3b-receptor (CR4) activity resembling CR3. J Clin Invest 82, 640-651.

Neefjes, J.J., Stollorz, V., Peters, P.J., Geuze, H.J., Ploegh, H.L., 1990. The biosynthetic pathway of MHC class II but not class I molecules intersects the endocytic route. Cell 61, 171-183.

Ney, P.A., D'Andrea, A.D., 2000. Friend erythroleukemia revisited. Blood 96, 3675-3680.

References

Nilsson, S.C., Sim, R.B., Lea, S.M., Fremeaux-Bacchi, V., Blom, A.M., 2011. Complement factor I in health and disease. Mol Immunol 48, 1611-1620.

Noris, M., Remuzzi, G., 2013. Overview of complement activation and regulation. Semin Nephrol 33, 479-492.

Oda, T., Kojima, Y., Akaike, T., Ijiri, S., Molla, A., Maeda, H., 1990. Inactivation of chemotactic activity of C5a by the serratial 56-kilodalton protease. Infect Immun 58, 1269- 1272.

Peng, Q., Li, K., Patel, H., Sacks, S.H., Zhou, W., 2006. Dendritic cell synthesis of C3 is required for full T cell activation and development of a Th1 phenotype. J Immunol 176, 3330- 3341.

Persons, D.A., Paulson, R.F., Loyd, M.R., Herley, M.T., Bodner, S.M., Bernstein, A., Correll, P.H., Ney, P.A., 1999. Fv2 encodes a truncated form of the Stk receptor tyrosine kinase. Nat Genet 23, 159-165.

Pickering, M.C., Cook, H.T., 2008. Translational mini-review series on complement factor H: renal diseases associated with complement factor H: novel insights from humans and animals. Clin Exp Immunol 151, 210-230.

Pincus, T., Rowe, W.P., Lilly, F., 1971. A major genetic locus affecting resistance to infection with murine leukemia viruses. II. Apparent identity to a major locus described for resistance to friend murine leukemia virus. J Exp Med 133, 1234-1241.

Posch, W., Steger, M., Knackmuss, U., Blatzer, M., Baldauf, H.M., Doppler, W., White, T.E., Hortnagl, P., Diaz-Griffero, F., Lass-Florl, C., Hackl, H., Moris, A., Keppler, O.T., Wilflingseder, D., 2015. Complement-Opsonized HIV-1 Overcomes Restriction in Dendritic Cells. PLoS Pathog 11, e1005005.

Power, C., 2001. Retroviral diseases of the nervous system: pathogenic host response or viral gene-mediated neurovirulence? Trends Neurosci 24, 162-169.

Preissner, K.T., Seiffert, D., 1998. Role of vitronectin and its receptors in haemostasis and vascular remodeling. Thromb Res 89, 1-21.

Probst, H.C., van den Broek, M., 2005. Priming of CTLs by lymphocytic choriomeningitis virus depends on dendritic cells. J Immunol 174, 3920-3924.

References

Pruenster, M., Wilflingseder, D., Banki, Z., Ammann, C.G., Muellauer, B., Meyer, M., Speth, C., Dierich, M.P., Stoiber, H., 2005. C-type lectin-independent interaction of complement opsonized HIV with monocyte-derived dendritic cells. Eur J Immunol 35, 2691-2698.

Racine, R., Chatterjee, M., Winslow, G.M., 2008. CD11c expression identifies a population of extrafollicular antigen-specific splenic plasmablasts responsible for CD4 T-independent antibody responses during intracellular bacterial infection. J Immunol 181, 1375-1385.

Raikow, R.B., JP, O.K., Buffo, M.J., Kociban, D.L., 1985. Effect of cyclophosphamide on Friend virus leukemogenesis in virus-sensitive and virus-resistant mice. Cancer Res 45, 555- 557.

Rein, A., 2011. Murine leukemia viruses: objects and organisms. Adv Virol 2011, 403419.

Ricklin, D., Hajishengallis, G., Yang, K., Lambris, J.D., 2010. Complement: a key system for immune surveillance and homeostasis. Nat Immunol 11, 785-797.

Robertson, M.N., Miyazawa, M., Mori, S., Caughey, B., Evans, L.H., Hayes, S.F., Chesebro, B., 1991. Production of monoclonal antibodies reactive with a denatured form of the Friend murine leukemia virus gp70 envelope protein: use in a focal infectivity assay, immunohistochemical studies, electron microscopy and western blotting. J Virol Methods 34, 255-271.

Robertson, M.N., Spangrude, G.J., Hasenkrug, K., Perry, L., Nishio, J., Wehrly, K., Chesebro, B., 1992. Role and specificity of T-cell subsets in spontaneous recovery from Friend virus- induced leukemia in mice. J Virol 66, 3271-3277.

Rock, K.L., Shen, L., 2005. Cross-presentation: underlying mechanisms and role in immune surveillance. Immunol Rev 207, 166-183.

Rooijakkers, S.H., Ruyken, M., Roos, A., Daha, M.R., Presanis, J.S., Sim, R.B., van Wamel, W.J., van Kessel, K.P., van Strijp, J.A., 2005. Immune evasion by a staphylococcal complement inhibitor that acts on C3 convertases. Nat Immunol 6, 920-927.

Rooijakkers, S.H., van Strijp, J.A., 2007. Bacterial complement evasion. Mol Immunol 44, 23-32.

Rus, H., Cudrici, C., Niculescu, F., 2005. The role of the complement system in innate immunity. Immunol Res 33, 103-112.

References

Sanchez-Madrid, F., Nagy, J.A., Robbins, E., Simon, P., Springer, T.A., 1983. A human leukocyte differentiation antigen family with distinct alpha-subunits and a common beta- subunit: the lymphocyte function-associated antigen (LFA-1), the C3bi complement receptor (OKM1/Mac-1), and the p150,95 molecule. J Exp Med 158, 1785-1803.

Saxena, S.K., Chitti, S.V., 2016. Molecular Biology and Pathogenesis of Retroviruses, Advances in Molecular Retrovirology. InTechOpen, pp. 3-18.

Schultz, K.R., Klarnet, J.P., Gieni, R.S., HayGlass, K.T., Greenberg, P.D., 1990. The role of B cells for in vivo T cell responses to a Friend virus-induced leukemia. Science 249, 921-923.

Schwartzberg, P., Colicelli, J., Goff, S.P., 1983. Deletion mutants of Moloney murine leukemia virus which lack glycosylated gag protein are replication competent. J Virol 46, 538-546.

Schwarz, M., Spath, L., Lux, C.A., Paprotka, K., Torzewski, M., Dersch, K., Koch-Brandt, C., Husmann, M., Bhakdi, S., 2008. Potential protective role of apoprotein J (clusterin) in atherogenesis: binding to enzymatically modified low-density lipoprotein reduces fatty acid- mediated cytotoxicity. Thromb Haemost 100, 110-118.

Sharma, A.K., Pangburn, M.K., 1996. Identification of three physically and functionally distinct binding sites for C3b in human complement factor H by deletion mutagenesis. Proc Natl Acad Sci U S A 93, 10996-11001.

Showers, M.O., DeMartino, J.C., Saito, Y., D'Andrea, A.D., 1993. Fusion of the erythropoietin receptor and the Friend spleen focus-forming virus gp55 glycoprotein transforms a factor-dependent hematopoietic cell line. Mol Cell Biol 13, 739-748.

Silver, J.E., Fredrickson, T.N., 1983. A new gene that controls the type of leukemia induced by Friend murine leukemia virus. J Exp Med 158, 493-505.

Sim, R.B., Day, A.J., Moffatt, B.E., Fontaine, M., 1993. Complement factor I and cofactors in control of complement system convertase enzymes. Methods Enzymol 223, 13-35.

Simon, V., Bloch, N., Landau, N.R., 2015. Intrinsic host restrictions to HIV-1 and mechanisms of viral escape. Nat Immunol 16, 546-553.

References

Singh-Jasuja, H., Thiolat, A., Ribon, M., Boissier, M.C., Bessis, N., Rammensee, H.G., Decker, P., 2013. The mouse dendritic cell marker CD11c is down-regulated upon cell activation through Toll-like receptor triggering. Immunobiology 218, 28-39.

Smith, D.S., Guo, K., Barrett, B.S., Heilman, K.J., Evans, L.H., Hasenkrug, K.J., Greene, W.C., Santiago, M.L., 2011. Noninfectious retrovirus particles drive the APOBEC3/Rfv3 dependent neutralizing antibody response. PLoS Pathog 7, e1002284.

Speth, C., Kacani, L., Dierich, M.P., 1997. Complement receptors in HIV infection. Immunol Rev 159, 49-67.

Stacker, S.A., Springer, T.A., 1991. Leukocyte integrin P150,95 (CD11c/CD18) functions as an adhesion molecule binding to a counter-receptor on stimulated endothelium. J Immunol 146, 648-655.

Starck, J., Doubeikovski, A., Sarrazin, S., Gonnet, C., Rao, G., Skoultchi, A., Godet, J., Dusanter-Fourt, I., Morle, F., 1999. Spi-1/PU.1 is a positive regulator of the Fli-1 gene involved in inhibition of erythroid differentiation in friend erythroleukemic cell lines. Mol Cell Biol 19, 121-135.

Steinman, R.M., Adams, J.C., Cohn, Z.A., 1975. Identification of a novel cell type in peripheral lymphoid organs of mice. IV. Identification and distribution in mouse spleen. J Exp Med 141, 804-820.

Steinman, R.M., Cohn, Z.A., 1973. Identification of a novel cell type in peripheral lymphoid organs of mice. I. Morphology, quantitation, tissue distribution. J Exp Med 137, 1142-1162.

Steinman, R.M., Cohn, Z.A., 1974. Identification of a novel cell type in peripheral lymphoid organs of mice. II. Functional properties in vitro. J Exp Med 139, 380-397.

Steinman, R.M., Hemmi, H., 2006. Dendritic cells: translating innate to adaptive immunity. Curr Top Microbiol Immunol 311, 17-58.

Steinman, R.M., Lustig, D.S., Cohn, Z.A., 1974. Identification of a novel cell type in peripheral lymphoid organs of mice. 3. Functional properties in vivo. J Exp Med 139, 1431- 1445.

Stoiber, H., Banki, Z., Wilflingseder, D., Dierich, M.P., 2008a. Complement-HIV interactions during all steps of viral pathogenesis. Vaccine 26, 3046-3054.

References

Stoiber, H., Frank, I., Spruth, M., Schwendinger, M., Mullauer, B., Windisch, J.M., Schneider, R., Katinger, H., Ando, I., Dierich, M.P., 1997. Inhibition of HIV-1 infection in vitro by monoclonal antibodies to the complement receptor type 3 (CR3): an accessory role for CR3 during virus entry? Mol Immunol 34, 855-863.

Stoiber, H., Soederholm, A., Wilflingseder, D., Gusenbauer, S., Hildgartner, A., Dierich, M.P., 2008b. Complement and antibodies: a dangerous liaison in HIV infection? Vaccine 26 Suppl 8, I79-85.

Stoiber, H., Speth, C., Dierich, M.P., 2003. Role of complement in the control of HIV dynamics and pathogenesis. Vaccine 21 Suppl 2, S77-82.

Strainic, M.G., Liu, J., Huang, D., An, F., Lalli, P.N., Muqim, N., Shapiro, V.S., Dubyak, G.R., Heeger, P.S., Medof, M.E., 2008. Locally produced complement fragments C5a and C3a provide both costimulatory and survival signals to naive CD4+ T cells. Immunity 28, 425-435.

Sunyer, J.O., Zarkadis, I.K., Lambris, J.D., 1998. Complement diversity: a mechanism for generating immune diversity? Immunol Today 19, 519-523.

Suresh, M., Molina, H., Salvato, M.S., Mastellos, D., Lambris, J.D., Sandor, M., 2003. Complement component 3 is required for optimal expansion of CD8 T cells during a systemic viral infection. J Immunol 170, 788-794.

Tacken, P.J., Figdor, C.G., 2011. Targeted antigen delivery and activation of dendritic cells in vivo: steps towards cost effective vaccines. Semin Immunol 23, 12-20.

Takeda, E., Tsuji-Kawahara, S., Sakamoto, M., Langlois, M.A., Neuberger, M.S., Rada, C., Miyazawa, M., 2008. Mouse APOBEC3 restricts friend leukemia virus infection and pathogenesis in vivo. J Virol 82, 10998-11008. te Velde, A.A., Keizer, G.D., Figdor, C.G., 1987. Differential function of LFA-1 family molecules (CD11 and CD18) in adhesion of human monocytes to melanoma and endothelial cells. Immunology 61, 261-267.

Tegla, C.A., Cudrici, C., Patel, S., Trippe, R., 3rd, Rus, V., Niculescu, F., Rus, H., 2011. Membrane attack by complement: the assembly and biology of terminal complement complexes. Immunol Res 51, 45-60.

References

Thieblemont, N., Haeffner-Cavaillon, N., Haeffner, A., Cholley, B., Weiss, L., Kazatchkine, M.D., 1995. Triggering of complement receptors CR1 (CD35) and CR3 (CD11b/CD18) induces nuclear translocation of NF-kappa B (p50/p65) in human monocytes and enhances viral replication in HIV-infected monocytic cells. J Immunol 155, 4861-4867.

Thornton, B.P., Vetvicka, V., Pitman, M., Goldman, R.C., Ross, G.D., 1996. Analysis of the sugar specificity and molecular location of the beta-glucan-binding lectin site of complement receptor type 3 (CD11b/CD18). J Immunol 156, 1235-1246.

Tjomsland, V., Ellegard, R., Che, K., Hinkula, J., Lifson, J.D., Larsson, M., 2011. Complement opsonization of HIV-1 enhances the uptake by dendritic cells and involves the endocytic lectin and integrin receptor families. PLoS One 6, e23542.

Turnberg, D., Botto, M., 2003. The regulation of the complement system: insights from genetically-engineered mice. Mol Immunol 40, 145-153.

Ueda, T., Rieu, P., Brayer, J., Arnaout, M.A., 1994. Identification of the complement iC3b binding site in the beta 2 integrin CR3 (CD11b/CD18). Proc Natl Acad Sci U S A 91, 10680- 10684. van der Touw, W., Cravedi, P., Kwan, W.H., Paz-Artal, E., Merad, M., Heeger, P.S., 2013. Cutting edge: Receptors for C3a and C5a modulate stability of alloantigen-reactive induced regulatory T cells. J Immunol 190, 5921-5925.

Vetvicka, V., Thornton, B.P., Ross, G.D., 1996. Soluble beta-glucan polysaccharide binding to the lectin site of neutrophil or natural killer cell complement receptor type 3 (CD11b/CD18) generates a primed state of the receptor capable of mediating cytotoxicity of iC3b-opsonized target cells. J Clin Invest 98, 50-61.

Villadangos, J.A., Schnorrer, P., 2007. Intrinsic and cooperative antigen-presenting functions of dendritic-cell subsets in vivo. Nat Rev Immunol 7, 543-555.

Vinay, D.S., Kwon, B.S., 2010. CD11c+CD8+ T cells: two-faced adaptive immune regulators. Cell Immunol 264, 18-22.

Walport, M.J., 2001a. Complement. First of two parts. N Engl J Med 344, 1058-1066.

Walport, M.J., 2001b. Complement. Second of two parts. N Engl J Med 344, 1140-1144.

References

Wang, F., St Gelais, C., de Silva, S., Zhang, H., Geng, Y., Shepard, C., Kim, B., Yount, J.S., Wu, L., 2016. Phosphorylation of mouse SAMHD1 regulates its restriction of human immunodeficiency virus type 1 infection, but not murine leukemia virus infection. Virology 487, 273-284.

Wilflingseder, D., Banki, Z., Dierich, M.P., Stoiber, H., 2005. Mechanisms promoting dendritic cell-mediated transmission of HIV. Mol Immunol 42, 229-237.

Wilflingseder, D., Banki, Z., Garcia, E., Pruenster, M., Pfister, G., Muellauer, B., Nikolic, D.S., Gassner, C., Ammann, C.G., Dierich, M.P., Piguet, V., Stoiber, H., 2007. IgG opsonization of HIV impedes provirus formation in and infection of dendritic cells and subsequent long-term transfer to T cells. J Immunol 178, 7840-7848.

Wilson, N.S., Villadangos, J.A., 2005. Regulation of antigen presentation and cross- presentation in the dendritic cell network: facts, hypothesis, and immunological implications. Adv Immunol 86, 241-305.

Wittmann, S., Behrendt, R., Eissmann, K., Volkmann, B., Thomas, D., Ebert, T., Cribier, A., Benkirane, M., Hornung, V., Bouzas, N.F., Gramberg, T., 2015. Phosphorylation of murine SAMHD1 regulates its antiretroviral activity. Retrovirology 12, 103.

Wong, W.W., Cahill, J.M., Rosen, M.D., Kennedy, C.A., Bonaccio, E.T., Morris, M.J., Wilson, J.G., Klickstein, L.B., Fearon, D.T., 1989. Structure of the human CR1 gene. Molecular basis of the structural and quantitative polymorphisms and identification of a new CR1-like allele. J Exp Med 169, 847-863.

Xie, C., Zhu, J., Chen, X., Mi, L., Nishida, N., Springer, T.A., 2010. Structure of an integrin with an alphaI domain, complement receptor type 4. EMBO J 29, 666-679.

Yeager, M., Wilson-Kubalek, E.M., Weiner, S.G., Brown, P.O., Rein, A., 1998. Supramolecular organization of immature and mature murine leukemia virus revealed by electron cryo-microscopy: implications for retroviral assembly mechanisms. Proc Natl Acad Sci U S A 95, 7299-7304.

Yoshimura, A., D'Andrea, A.D., Lodish, H.F., 1990. Friend spleen focus-forming virus glycoprotein gp55 interacts with the erythropoietin receptor in the endoplasmic reticulum and affects receptor metabolism. Proc Natl Acad Sci U S A 87, 4139-4143.

References

Zelinskyy, G., Robertson, S.J., Schimmer, S., Messer, R.J., Hasenkrug, K.J., Dittmer, U., 2005. CD8+ T-cell dysfunction due to cytolytic granule deficiency in persistent Friend retrovirus infection. J Virol 79, 10619-10626.

Zhang, J.X., Diehl, G.E., Littman, D.R., 2008. Relief of preintegration inhibition and characterization of additional blocks for HIV replication in primary mouse T cells. PLoS One 3, e2035.

Zhang, W., Cao, S., Martin, J.L., Mueller, J.D., Mansky, L.M., 2015. Morphology and ultrastructure of retrovirus particles. AIMS Biophys 2, 343-369.

Zheng, Y.H., Jeang, K.T., Tokunaga, K., 2012. Host restriction factors in retroviral infection: promises in virus-host interaction. Retrovirology 9, 112.

Zipfel, P.F., Skerka, C., 1994. Complement factor H and related proteins: an expanding family of complement-regulatory proteins? Immunol Today 15, 121-126.

Zipfel, P.F., Skerka, C., 2009. Complement regulators and inhibitory proteins. Nat Rev Immunol 9, 729-740.

Zon, L.I., Moreau, J.F., Koo, J.W., Mathey-Prevot, B., D'Andrea, A.D., 1992. The erythropoietin receptor transmembrane region is necessary for activation by the Friend spleen focus-forming virus gp55 glycoprotein. Mol Cell Biol 12, 2949-2957.

Curriculum Vitae

CURRICULUM VITAE

Personal Data

Name Muneeb Ahmad IDREES Date of Birth 03.08.1984 Nationality Pakistani Marital Status: Married Email [email protected], [email protected]

Education

Since March 2013 Doctor of Philosophy Department of Hygiene, Microbiology and Social Medicine, Division of Virology, Medical University Innsbruck Thesis Title: “Role of complement receptors in the induction of retroviral-specific CTL responses by dendritic cells” Supervisor: Prof. Dr. Heribert Stoiber

March 2007 – July 2009 Master of Philosophy (M.Phil) in Pharmaceutics Department of Pharmacy, The Islamia University of Bahawalpur, Pakistan Thesis Title: “Formulation and evaluation of microemulsion containing flurbiprofen for topical delivery” Supervisor: Prof. Dr. Nisar-ur-Rahman

Dec 2002 – Dec 2006 B. Pharmacy Department of Pharmacy, The Islamia University of Bahawalpur, Pakistan

Curriculum Vitae

Professional Experience

Aug 2009 – Feb 2013 Lecturer Department of Pharmacy, COMSATS Institute of Information Technology, Abbottabad, Pakistan

March 2010 – July 2010 Research trainee in the project “Microfluidic chip- based system for high-throughput enzyme screening” Dalian Institute of Chemical Physics, Chinese Academy of Sciences, China (Was sent for training by COMSATS IIT for lab up- gradation)

Feb 2007 – March 2007 Internee Production Pharmacist Stiefel Laboratories Pakistan (Pvt) Ltd., Pakistan

Awards and Honors

March 2013 – Feb 2017 Higher Education Commission Pakistan-ÖAD funded Overseas PhD scholarship

March 2007 – Feb 2009 Merit-based District Education Scholarship during M.Phil

Dec 2002 – Dec 2006 Merit-based District Education Scholarship during B. Pharmacy

 Completed M.Phil thesis with distinction and secured 1st position in the Department of Pharmacy

Research Publications

1. Leonaviciute G, Bonengel S, Mahmood A, Idrees MA, Bernkop SA. “S-protected thiolated hydroxyethyl cellulose (HEC): Novelmucoadhesive excipient with improved stability”. Carbohydr Polym. 144: 514–521; 2016.

Curriculum Vitae

2. Mahmood A, Bonengel S, Laffleur F, Ijaz M, Idrees MA, Hussain S, Huck CW, Matuszczak B, Bernkop SA. “Can thiolation render a low molecular weight polymer of just 20-kDa mucoadhesive?”. Drug Dev Ind Pharm. 42 (5): 686-93; 2016. 3. Laffleur F, Röggla J, Idrees MA, Griessinger J. “Chemical Modification of Hyaluronic Acid for Intraoral Application”. J Pharm Sci. 103 (8): 2414-23; 2014. 4. Mohsin S, Rahman NU, Idrees MA, Sarfraz MK, Khan MK, Mustafa G. “Suitability of Gelucire 50/13 for controlled release formulation of salbutamol sulphate”. Pak J Pharm Sci. 25 (1): 35-41; 2012. 5. Idrees MA, Rahman NU, Ahmad S, Ali MY, Ahmad I. “Enhanced transdermal delivery of flurbiprofen via microemulsions: Effects of different types of surfactants and cosurfactants”. DARU. 19 (6): 433-439; 2011. 6. Ali Y, Rahman NU, Idrees MA, Mohsin S, Ahmed S, Ahmed I. “Sustained Release of Captopril from Matrix Tablet Using Methylcellulose in a New Derivative Form”. Lat Am J Pharm. 30 (9): 1696-1701; 2011. 7. Shah SMA, Malik NS, Awan A, Idrees MA, Akram M, Asif HM, Nawaz S. “Prevalence of amoebiasis in Gadap Town, Karachi”. Afr J Biotechnol. 10 (54): 11214-11216; 2011.

Acknowledgements

ACKNOWLEDGEMENTS

This acknowledgement is not merely a manifestation of formal duty, but all the emotional associations I have with the persons who helped me during my work and made this thesis presentable. From the formative stages of this work, to the final draft, I owe a profound debt of gratitude to my supervisor, Prof. Dr. Heribert Stoiber, for his hard work, guidance, unfailing supportive attitude and sound advice throughout the entire PhD and for believing in my abilities; showing me places where I had strayed from the straight path and gently leading me back in the proper direction. His inspiring help, consistent encouragement and affectionate attitude during the entire study duration will ever be remembered. I have learned so much, and without you, this would not have been possible. Thank you so much for a great experience.

I express my deep indebtedness and thanks to Dr. Zoltan Banki, the most engaged co- supervisor there could be; whose inspiring attitude leads to higher ideas of research work. I gratefully acknowledge his supervision, advice and critical contribution towards this work.

Deepest thanks are due to Mag. Brigitte Müllauer for introducing me into all the different techniques and for always giving a caring and helping hand.

Many thanks go to Britta Schiela for giving me such a pleasant time and creating a friendly atmosphere during work and at the office; you more than once made my day by making me laugh. I would like to extend my heartfelt gratitude to my colleagues Daniela Bichler (who became Daniela Krenn a couple of days ago; my heartiest congratulations) and Iris Koske for helping me a lot during my work and creating such a great friendship at the office. I cannot forget to mention Daniela Deutschmann and Simon Kruis, I am especially happy that we had an opportunity to work together. All my colleagues contributed to make the lab a pleasant and amusing place to work at; you have all made it a memorable time of my life. I will miss you; good luck to each of you in your future endeavors.

Last but not least, I wish to express my thanks to all my family members especially my parents, siblings, wife and son. I owe my success to their moral support, spiritual guidance and special care for me. Their day and night sincere prayers carved my way to success. My son, Umar Ahmad Idrees, who joined us during my PhD, brought to me boundless pleasure and happiness and made me relax during the hard times. There is no way to express my appreciation to my wife Sadia who unselfishly supported, encouraged, loved, entertained and helped me with patience to get through this agonizing period in the most positive way.

Acknowledgements

I am also thankful to the Higher Education Commission of Pakistan (HEC) and Austrian Agency for International Co-operation in Education and Research (ÖAD) for the funding.