Trafficking of Fcγriia and Fcγriib2 Upon Endocytosis of Immune Complexes

Trafficking of FcγRIIA and FcγRIIB2 upon Endocytosis of Immune Complexes

Christine Yue Zhang

A thesis submitted in conformity with requirements For the degree of Doctor of Philosophy

Graduate Department of Immunology University of Toronto

© Copyright by Christine Yue Zhang (2011) Trafficking of FcγRIIA and FcγRIIB2 upon Endocytosis of Immune Complexes Christine Yue Zhang Doctor of Philosophy, 2011 Graduate Department of Immunology University of Toronto

Abstract

Fcγ receptors (FcγR) which recognize the Fc fraction of IgG play key roles in the modulation of a range of cellular responses as part of the host defense against foreign microbes and antigens.

An important function of FcγR is to mediate internalization of soluble IgG-containing immune complexes via endocytosis. The mechanisms of internalization and intracellular transport of

FcγR after internalization are less clear. In this thesis, I investigated the trafficking behaviours of human FcγRIIA and FcγRIIB2 upon clustering with immune complexes. In Chapter 3, I demonstrate FcγRIIA, when engaged with multivalent heat aggregated IgG (agIgG), is delivered along with its ligand to lysosomal compartments for degradation, whereas FcγRIIB2 becomes dissociated from the ligand and routed separately into a recycling pathway. FcγRIIA sorting to lysosomes requires receptor multimerization, but does not require either Src family kinase (SFK) activity or receptor ubiquitylation. Upon co-engagement, these two receptors are sorted independently to distinct final fates after dissociating from their co-clustering ligand. In

Chapter 4, I show that while the ubiquitin-conjugating system is required for FcγRIIA-mediated endocytosis, it is not required for FcγRIIB2 endocytosis. FcγRIIB2 internalizes immune complexes at a faster rate than FcγRIIA and accelerates the endocytosis of FcγRIIA upon receptor co-engagement. Taken together, these results reveal fundamental differences in the trafficking behaviour of FcγRIIA and FcγRIIB2 both during the initial induction of endocytosis as well as during subsequent intracellular sorting.

Acknowledgements

Upon the completion of this PhD thesis, I would first like to give a big thank you to my supervisor, Dr. James Booth, for providing me the opportunity to work on this project and for providing me tremendous support throughout my PhD study. Dr. Booth was the one who first led me to this exciting field of biomedical research, as the years go by, my enthusiasm remains.

With his guidance, I have acquired more than just the necessary skills for conducting successful experimental studies. I have also learned the essential qualities of being a good researcher, which will be valuable assets for my future career path.

Many thanks to my supervisory committee members, Dr. Tania Watts and Dr. David

Williams, for all of their advices and supports. I enjoyed every committee meeting and really appreciate all the time they have given me. This project would not have been possible without their help.

Moreover, I would like to thank every current and previous members of the Booth lab, especially Courtney McIntosh and Patricia Mero, for all of their technical supports and for making the lab environment such an enjoyable place to be. They have showed me teamwork is just as important as independent working capability to the success of research study.

My thanks also go to the Department of Immunology at the University of Toronto which has made my undergraduate and graduate studies such a memorable experience.

Last but not least, to my parents, thank you so much for all these years of support. I would not have gone this far without them.

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Table of Contents

Abstract…………………………………………………………..……………………………..ii

Acknowledgements…………………………………………………..………………………...iii

Table of Contents……………………………………………..………………………………..iv

List of Tables…………………………………..………………………...…………………….vii

List of Figures..…………………………………………………………...…………………..viii

List of Abbreviations……………………………………………………...…………………..xi

List of Publications…………………………………………………..………….………..….xiii

Chapter 1 Introduction……………………….……………………………………………….1

1.1 Fcγ receptors general background……………………………………………………...1

1.2 FcγRIIA and FcγRIIB2………………………………………………………………....7

1.3 Binding affinity of FcγRs to IgG subclasses…………………………..……………...13

1.4 Endocytosis……………………………………………………………………………15

1.5 Intracellular trafficking and antigen presentation of FcγR………………………..…..21

1.6 Receptor ubiquitylation and phosphorylation in FcγR-mediated endocytosis………..23

1.7 Project aims and thesis outline………………………………………….………….....31

Chapter 2 Materials and Methods…………………………………….…………………….35

2.1 Reagents and antibodies………………………………………………………….…...35

2.2 Cell culture………………………………………………………….………………...36

2.3 DNA constructs and transfection………..…………...………………..………………37

2.4 Ubiquitylation abolishment and inhibitor treatment…………………..………………39

2.5 Development of rabbit polyclonal antibodies specific to the intracellular domains of FcγRIIA or FcγRIIB2…………………...…………..……………………….……..39

2.6 Endocytosis and phagocytosis assays………….………………………………………40

2.7 Microscopy and immunofluorescence…………………………………….……….….41

2.8 Flow cytometry……………………………………………………………….….…....42

2.9 Immunoprecipitation and Western Blotting………………………………….….……43

Chapter 3 Divergent Intracellular Sorting of FcγRIIA and FcγRIIB2………….…...... 45

3.1 Abstract………………………………………………….………………….……….45

3.2 Introduction……………………………………………………….……….…...…....46

3.3 Results…………………………………………………………………………….....48

3.3.1 Aggregated IgG internalized by either FcγRIIA or FcγRIIB2 is degraded in lysosomes………………………………………………….…….……...48

3.3.2 FcγRIIA, but not FcγRIIB2, undergoes degradation in lysosomes after internalization………………………………………………………...... ….48

3.3.3 Extracellular domains of FcγRIIA and FcγRIIB2 do not determine their differential trafficking…………………………..………………...………..60

3.3.4 Sorting of FcγRIIA and FcγRIIB2 upon persistent cross-linking with antibody complexes………………………………………………..….……60

3.3.5 FcγRIIA dimerization induces receptor tyrosine phosphorylation…...……..66

3.3.6 Sorting of FcγRIIA and FcγRIIB2 in primary human myeloid cells...……...66

3.3.7 FcγRIIA sorting for degradation does not require Src family kinase activity or receptor ubiquitylation at lysine residues …………...……..…..69

3.3.8 FcγRIIA and FcγRIIB2 trafficking upon co-engagement……………...……80

3.4 Discussion…………………………………………..………………………….……87

3.5 Appendix..…………………………………………….…..………………………….……92

Chapter 4 Comparison of Endocytosis Mediated by FcγRIIA and FcγRIIB2……...... 96

4.1 Abstract………………………………………………………………………….…..96

4.2 Introduction……………………………………………………………..…….……..97

4.3 Results……………………………………………………………..…………….…..99

4.3.1 Identification of a region with the ITAM of FcγRIIA crucial for its endocytosis……………………………………………………...……….....99

4.3.2 Endocytosis of FcγRIIB2 does not require ubiquitylation…………..….….101

4.3.3 FcγRIIA, but not FcγRIIB2, is ubiquitylated upon receptor engagement....103

4.3.4 Rate of endocytosis mediated by FcγRIIA and FcγRIIB2…………..…...... 103

4.3.5 Acceleration of FcγRIIA internalization by FcγRIIB2…………….….…...109

4.3.6 FcγRIIB2 upregulation results in an increase in the rate of FcγRII internalization in monocytes……………..………………….……..……..109

4.4 Discussion……………………………………………………………….…………111

Chapter 5 General Discussin and Conclusion..…………….…………………………..….117

5.1 Thesis overview….…………………………………………………………………117

5.2 Characterization of FcγRIIA and FcγRIIB2 trafficking in primary human myeloid lineage cells…………………………..…………………..…………...…121

5.2.1 The complexity of human myeloid cells--various DC and macrophage subsets………………………………………..…………………………...121

5.2.2 Challenges of studying FcγRII trafficking in primary human myeloid cells……………………………………………………………………….122

5.2.3 Future Plan…………………………..……………………………………..125

5.3 Identification of E3 ligase and ubiquitylation sites for FcγRIIA ubiquitylation ….126

5.4 Association of FcγRIIA trafficking and signaling………………...……………….128

5.5 Effect of Trafficking of FcγRIIA and FcγRIIB2 on Antigen Presentation….….….130

5.6 Clinical implications………………………………………………….……………132

5.7 Conclusions……………………………………………………………...…………133

References……………………………………………………………………………….……135

List of Tables

Table 1-1 Summary of research findings on the expression of FcγRIIA and FcγRIIB and the effects of cytokine regulations in primary human myeloid cells………………..…..12

Table 1-2 Binding of IgG isotypes to FcγRs…………………………………………………...15

Table 3-1: The expression of FcγRIIA and FcγRIIB in various human myeloid cells………..68

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List of Figures

Fig. 1-1 FcγR family in human and mouse ……………………………………………..……...3

Fig. 1-2 Signaling pathways of activating and inhibiting FcγRs triggered upon immune complex binding…………………………………………………………………..…...4

Fig. 1-3 Human FcγRII subfamily………………………………………………………...... ….8

Fig. 1-4 Endosomal compartments and the trafficking of cell surface receptors……..……….17

Fig. 1-5 Ubiquitylation process of target protein………………………………………….…..25

Fig. 1-6 The ESCRT complexes in MVB sorting of EGFR……………………….….….……29

Fig. 3-1 AgIgG internalized by either FcγRIIA or FcγRIIB2 is degraded in lysosomes....…...49

Fig. 3-2 FcγRIIA, but not FcγRIIB2, undergoes degradation after internalization…….…...…51

Fig. 3-3 The effect of cycloheximide on the level of FcγRIIB2 with or without ligand stimulation……………………………………………………………………..……...53

Fig. 3-4 MH tag does not affect the intracellular sorting of FcγRIIA- or FcγRIIB2-mediated endocytosis………...……………...... 54

Fig. 3-5 Rabbit anti-FcγRIIA sera and anti-FcγRIIB sera…….………………………………..55

Fig. 3-6 FcγRIIA, but not FcγRIIB2, follows agIgG to lysosomes………………...………….57

Fig. 3-7 FcγRIIB2, but not FcγRIIA is sorted into Rab11-positive recycling endosomes after internalization………………………………………………….…..…………….59

Fig. 3-8 Extracellular domains of FcγRIIA and FcγRIIB2 do not determine their differential trafficking………………………………………...…………………..…...61

Fig. 3-9 Sorting of FcγRIIB2 after engagement with anti-FcγRII antibodies………………….63

Fig. 3-10 FcγRIIA, but not FcγRIIB2, undergoes degradation after cross-linking via anti-FcγRII antibody complexes………………………….…………………..……...64

Fig. 3-11 Sorting of FcγRIIA after engagement with anti-FcγRIIA antibodies………………..65

Fig. 3-12 ITAM-mediated tyrosine phosphorylation of FcγRIIA with different degree of cross-linking……………………………..…………..………………..……………..67

Fig. 3-13 Antibody complexes internalized by FcγRIIA are first sorted into EEA1+ early endosomes in human macrophages…………..……………………………..……….70

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Fig. 3-14 Antibody complexes internalized by FcγRIIA are sorted to lysosomal compartments in human macrophages…………………………………….….……..71

Fig. 3-15 The sorting of antibody complexes internalized by FcγRIIA to lysosomes in human monocytes and immature moDC……………………………..………….…….…….72

Fig. 3-16 FcγRIIA-mediated internalization of anti-FcγRIIA antibodies in immature moDC...73

Fig. 3-17 Src family kinase activity is not required for FcγRIIA sorting for degradation……..75

Fig. 3-18 Effect of PP1 and LY294002 on the kinetics of FcγRIIA degradation…….………..77

Fig. 3-19 Efficacy test of PP1 and LY294002…………………….…………………………...78

Fig. 3-20 Effect of PP1 on agIgG-induced FcγRIIA degradation in THP1……………..……...79

Fig. 3-21 Receptor lysine residues are not necessary for FcγRIIA degradation…….…………81

Fig. 3-22 FcγRIIA and FcγRIIB2 sorting upon co-engagement with agIgG…………..………82

Fig. 3-23 Expression of cotransfected GFP as a measure of transient receptor expression……85

Fig. 3-24 FcγRIIA and FcγRIIB2 sorting upon co-engagement with anti-FcγRII antibodies….86

Appendix Fig. 1 Microscopy analysis of co-localization of agIgG internalized by FcγRIIA to LAMP1…………………………………………..……………………..……………..94

Fig. 4-1 Identification of key residues in FcγRIIA intracellular domain crucial for the induction of its endocytosis…………………………………………..………...…...100

Fig. 4-2 Endocytosis of FcγRIIB2 does not require ubiquitylation………….……….………102

Fig. 4-3 MycHis tag does not affect the initial induction of FcγRIIA- or FcγRIIB2-mediated endocytosis…………………………………………………………….……….……102

Fig. 4-4 FcγRIIA but not FcγRIIB2 becomes ubiquitylated during the induction of endocytosis……………………………………………………………..…...……….104

Fig. 4-5 FcγRIIB2 endocytosis of agIgG is more rapid than that of FcγRIIA……….………105

Fig. 4-6 FcγRIIB2 endocytosis of antibody complexes is more rapid than that of FcγRIIA...107

Fig. 4-7 Surface ligands are not released to the medium after FcγRII engagement in warm medium……………………………………………..………………………………..108

Fig. 4-8 Acceleration of FcγRIIA internalization by FcγRIIB2……………...……………….110

Fig. 4-9 FcγRIIB upregulation in human monocytes results in an increase in the rate of internalization of cross-linked FcγRII………………………………….…….……..112

Fig. 5-1 Schematic representation of FcγRIIA and FcγRIIB2 trafficking when engaged or co- engaged with immune complexes…………………………………………………..118

Fig. 5-2 Two mechanisms of downregulation of FcγRIIA-mediated activating signals by FcγRIIB2………………………………………………………………..…………..120

List of Abbreviations

Ab antibody

ADCC antibody-dependent cellular cytotoxicity agIgG heat aggregated IgG

AP2 clathrin-associated adaptor protein 2

APC antigen presenting cells

BCR B cell receptor

BFM bafilomycin

CI-MRP cation-independent mannose-6-phospate receptor

Cbl Casitas B-lineage lymphoma

DC dendritic cells

EEA1 early endosome antigen 1

EGF/EGFR epidermal growth factor/epidermal growth factor receptor

ESCRT endosomal sorting complex required for transport

FcγR Fcγ receptor

GHR growth hormone receptor

Hrs Hepatocyte growth factor-regulated tyrosine kinase substrate iDC immature dendritic cells

Ig immunoglobulin

IIA FcγRIIA

IIB2 FcγRIIB2

IL- interleukin- immune-EM immune-electron microscopy

ITAM immunoreceptor tyrosine-based activation motif

ITIM immunoreceptor tyrosine-based inhibition motif

ITP thrombocytic purpura

IVIg intravenous immunoglobulin

K63/K48 lysine 63/lysine 48

LAMP1 lysosomal-associated membrane protein 1

LDL/LDLR low-density-lipoprotein/low-density-lipoprotine receptor

mDC mature dendritic cells

MFI mean fluorescence intensity

MH MycHis

MHC major histocompatibility complex

MIIC MHC class II containing compartments

MVB multivesicular body

moDC monocyte-derived dendritic cells

PBS/PBS-T phosphate buffered saline/ phosphate buffered saline-Tween

SFK Src family kinases

PI3K phosphatidylinositol 3-kinase

RING really interesting new gene

SHIP SH2-domain-containing inositol polyphosphate 5′ phosphatase

SHP1 SH2-domain-containing protein tyrosine phosphatase 1

SLE systemic lupus erythematosus

TLR toll-like receptor

TBS/TBS-T tris buffered saline/ tris buffered saline-Tween

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List of Publications

Zhang CY and Booth JW. 2010. Divergent Intracellular Sorting of FcγRIIA and FcγRIIB2. J Biol Chem. 285(44):34250-34258

Zhang CY and Booth JW. Comparison of Endocytosis Mediated by FcγRIIA and FcγRIIB2. 2010. Mol Immunol. submitted.

Mero P, Zhang CY, Huang ZY, Kim MK, Schreiber AD, Grinstein S, Booth JW. 2006. Phosphorylation-independent ubiquitylation and endocytosis of FcγRIIA. J Biol Chem. 281 (44): 33242-33249

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Chapter 1

Introduction

The aim of this thesis is to characterize the trafficking behaviours of two members of the

human Fcγ receptors (FcγRs), FcγRIIA and FcγRIIB2, upon engagement with immune

complexes for endocytosis. This chapter begins with an overview of the structural and

functional characteristics of different human FcγRs, with a particular emphasis on the

biology of FcγRIIA and FcγRIIB2. The clathrin-dependent endocytosis pathway and

various endocytic compartments are also reviewed. In addition, the intracellular transport

of Fc receptors and their ability to enhance antigen presentation are discussed. The last

section of the introduction gives an overview of ubiquitylation and its role in FcγR-

mediated endocytosis.

1.1 Fcγ receptors: general background

Fc receptors, a family of transmembrane proteins recognizing the Fc fraction of

immunoglobulin, are key players in the modulation of cellular responses triggered by immune complexes and in the host defense against foreign microbes. These receptors, widely expressed in a variety of cells in the hematopoietic system, can carry out a range of effector functions. These functions include antibody-dependent cellular cytotoxicity

(ADCC), release of oxygen metabolites and pro-inflammatory mediators, modulation of

B cell and dendritic cell (DC) activation, induction of mast cell degranulation, neutrophil activation as well as clearance of immune complexes from circulation (Nimmerjahn and

Ravetch, 2008). The uptake of immune complexes through FcγRs has also been shown to

increase antigen presentation efficiency by several orders of magnitude (Sallusto and

Lanzavecchia, 1994).

FcγRs, the subset of the Fc receptor family that bind to IgG, are present in both human and mouse. In humans and other primates, the FcγR family consists of multiple

members. This includes the high affinity IgG receptor FcγRI and the low affinity IgG receptors FcγRII (FcγRIIA, FcγRIIB1, FcγRIIB2, FcγRIIC) and FcγRIII (FcγRIIIA and

FcγRIIIB) (Fig. 1-1). The extracellular domains of these membrane bound glycoproteins are composed of 2-3 immunoglobulin-like domains. FcγRI with three Ig-like domains in its extracellular domains is the only member in the FcγR family that binds to monomeric

IgG with high affinity. Other low affinity FcγRs only interact with multimeric ligands such as immune complexes or antibody-opsonized bacteria. Receptor clustering is required for signaling activation for all FcγRs. Although extracellular domains of various low affinity FcγRs are similar in their general structure, they bind to different IgG subtypes with varying affinities and specificities (Takai, 2005).

FcγRs can be categorized into two groups based on the signaling motif each FcγR carries in its intracellular domain. The activating FcγRs (in humans, FcγRI, FcγRIIA and

FcγRIIIA) send activating signals through immunoreceptor tyrosine-based activation motifs (ITAMs) that lie either in the receptor itself or in the γ-chain associated with the receptor. Cross-linking of the activating FcγRs results in receptor phosphorylation at tyrosine residues in the ITAMs by Src family kinases (SFK) including Lyn, Fyn, Fgr and

Hck (Fig. 1-2A) (Bonnerot et al., 1997; Isakov, 1997; Nimmerjahn et al., 2008; Ravetch and Bolland, 2001). This leads to the recruitment of Syk to the phosphorylated receptors followed by the induction of downstream signaling cascades. One of these early

Fig. 1-1 FcγR family in human and mouse Schematic representation of the human FcγR family which is grouped into two categories: 1) the activating receptors including FcγRI (CD64), FcγRIIA (CD32A), FcγRIIC (CD32C) and FcγRIIIA (CD16A); 2) the inhibitory receptors including FcγRIIB1 and FcγRIIB2. FcγRIIIB (CD16B) is a GPI-anchored receptor without an intracellular domain. The activating receptors have immunoreceptor tyrosine-based activation motifs (ITAMs) either on the γ chain associated with the receptor α chain or on the receptor itself. They are capable of inducing downstream pro-inflammatory responses. The inhibitory receptors with immunoreceptor tyrosine-based inhibition motif s (ITIMs) in their cytoplasmic tails can downregulate effector responses induced by the activating receptors. The yellow arrow points to the FcγRIIB1-specific insertion that prevents coated pit formation and receptor internalization. The mouse activating FcγRs include FcγRI, FcγRIII and FcγRIV whereas FcγRIIB1 and FcγRIIB2 are the inhibitory FcγRs in mouse.

Fig. 1-2 Signaling pathways of activating and inhibiting FcγRs triggered upon immune complex binding (adapted from Nimmerjahn F, 2008) A) Upon clustering of activating FcγRIII with multivalent immune complexes, tyrosines of the ITAMs in the receptor-associated γ chain are phosphorylated by Src family kinases (SFK) such as Lyn. This leads to the recruitment of Syk which activates a number of signal-transducing molecules including phosphoinositide 3-kinase (PI3K) and son of sevenless homologue (SOS).

PI3K mediates the generation of phosphatidylinositol-3,4,5-trisphosphate (PI(3,4,5)P3) which activates phospholipase Cγ (PLCγ) and Bruton’s tyrosine kinase (BTK). As a result, a series of downstream signaling pathways are activated including the ERK, p38 and Jun pathways, triggering a range of inflammatory responses and calcium flux. B) Cross-linking of the activating FcγRs with the inhibitory FcγRIIB leads to the phosphorylation of ITIMs of FcγRIIB at tyrosine residues by SFK, resulting in the recruitment of phosphatases including SRC- homology-2-domain-containing inositol-5-phosphatase (SHIP) and SH2-domain-containing protein tyrosine phosphatase 1 (SHP1). The activating receptors are dephosphorylated and

PI(3,4,5)P3 is hydrolysed to phosphatidylinositol-3,4-bisphosphate (PI(3,4)P2), resulting in the inhibition of PLCγ and BTK recruitment and down-regulation of the activating signal.

signaling event is the activation of phosphoinositide 3-kinase (PI3K). Phosphatidylinositol-

3,4,5-trisphosphate (PI(3,4,5)P3) is generated as a result, leading to the activation of phospholipase Cγ (PLCγ) and Bruton’s tyrosine kinase (BTK). PLCγ is responsible for inducing secondary messengers inositol 1,4,5-triphosphate (IP3) and diacylglycerol (DAG), which are important for sustained calcium mobilization and activation of protein kinase C

(PKC), respectively. BTK triggers the RAC-RHO pathway. Son of sevenless homologue (SOS) activated by Syk triggers the RAS-RAF-MAPK pathway. The activation of downstream signaling molecules including ERK, p38 and JUN induces a number of cellular responses including ADCC, phagocytosis, cytokine production and oxidative burst.

The inhibitory receptors (FcγRIIB1 and FcγRIIB2) have signaling effects opposite to the activating receptors due to the immunoreceptor tyrosine-based inhibition motifs (ITIMs) in their intracellular domains. Co-engagement of FcγRIIB with other ITAM-containing receptors such as B cell receptors (BCR) results in tyrosine phosphorylation of the ITIM in FcγRIIB by

SFK (Fig. 1-2B). As a result, phosphatases are recruited including SRC-homology-2-domain- containing inositol-5-phosphatase (SHIP) and SH2-domain-containing protein tyrosine phosphatase 1 (SHP1) (Joshi et al., 2006; Nimmerjahn et al., 2008; Ono et al., 1996; Ono et al.,

1997; Ravetch et al., 2001). Phosphoinositide PI(3,4,5)P3 is hydrolysed, inhibiting the recruitment of BTK or PLCγ to the activating receptors and their downstream processes such as calcium mobilization, cytokine release and cellular proliferation.

The integration of both positive and negative signals by multiple FcγRs sets the threshold for cell activation and maintains a well-balanced overall immune response (Nimmerjahn et al.,

2008). This ensures successful clearance of IgG-bound foreign antigen and microbes while preventing the development of autoimmune diseases. Aberrant expression or allelic polymorphism of FcγRs resulting in alteration of their functionality and shift in the overall

activating and inhibitory signaling can contribute to the exacerbation of these diseases.

Genome-wide linkage studies in human systemic lupus erythematosus (SLE) have identified the linkage of susceptibility gene loci including various low affinity members in FcγR (FcγRIIA,

FcγRIIB, FcγRIIIA and FcγRIIIB) to the disease (Duits et al., 1995; Hatta et al., 1999; Koene et al., 1998; Kyogoku et al., 2002). FcγRs have also been implicated in other immune diseases such as rheumatoid arthritis (Nieto et al., 2000), inflammatory bowel disease (Weersma et al.,

2010) and multiple sclerosis (Myhr et al., 1999). Studies have been carried out in genetically engineered murine models to investigate FcγRs mechanisms in the development of human autoimmune diseases. Deletion of activating FcγRs or the associated γ-chain renders mice resistant to the induction or spontaneous onset of various autoimmune diseases (Barnes et al.,

2002; Hazenbos et al., 1996; Park et al., 1998). In contrast, mice deficient in the inhibitory

FcγRIIB show enhanced inflammatory responses in a range of disease models, indicating the important role of FcγRII in antagonizing positive signals (Takai, 2005).

Much effort has been put into the characterization of the opposite signalling effects of activating and inhibitory FcγRs. Extensive investigations have been carried out to understand the role FcγRs play as susceptibility factors in a range of autoimmune diseases. However, the mechanism of FcγRs trafficking within cells, including both the internalization and the subsequent processing of their ligands, remains less clear. It is important to investigate the fates of FcγRs and immune complexes in order to understand both the regulation of inflammatory signalling by FcγRs and the presentation of antigen internalized in immune complexes by antigen presenting cells (APCs). Some studies indicate that immune complexes internalized by FcγRs are degraded in the lysosomes, leading to their removal from the circulation. Other studies suggest that some FcγRs can recycle intact native antigen to the cell surface of dendritic cells (DCs) for direct presentation to the splenic marginal zone B cells

through antigen-BCR interaction (Bergtold et al., 2005; Harrison et al., 1994; Mellman and

Plutner, 1984).

1.2 FcγRIIA and FcγRIIB2

A unique feature of the human immune system that distinguishes it from that of the mouse is that human myeloid cells express both activating and inhibitory members of the

FcγRII subfamily, namely FcγRIIA, FcγRIIC, FcγRIIB1 and FcγRIIB2 (Fig. 1-3A) . FcγRIIA is an activating member of the Fc receptor family present only in humans and other primates.

This 40kDa glycoprotein is the most widely expressed FcγRs in humans and is present on various leukocytes including neutrophils, eosinophils, platelets, mast cells, macrophages, monocytes, Langerhans cells and DCs (Tan Sardjono et al., 2003; Zhao et al., 2006). In human, chimpanzee and mouse, FcγRII genes are located on chromosome 1 while these genes are mapped to different chromosomes in other species (Nimmerjahn and Ravetch, 2006; Qiu et al.,

1990; Sammartino et al., 1988).

The structure of FcγRIIA is distinct from the activating FcγRI and FcγRIII as it is a single-chain FcγR that does not require the participation of Fc receptor γ-chain to trigger the downstream signalling cascades. The pro-inflammatory signalling of FcγRIIA is triggered through an ITAM domain that lies within its intracellular tail. This motif is similar but not identical to the ITAM in Fc receptor γ-chain, with a unique sequence of 12 instead of 7 amino acids in between the two YxxL motifs (Fig. 1-3B). The aggregation of FcγRIIA by multivalent ligands such as immune complexes results in receptor phosphorylation by SFK and the activation of downstream signalling pathways. Another unique characteristic of FcγRIIA is its ability to form dimers with other activating FcγRs including FcγRIIIB and FcγRI and to act in synergy during cell activation (Daeron, 1997; Tan Sardjono et al., 2003). The ITAM of

Fig. 1-3 Human FcγRII subfamily A) Schematic representation of the human FcγRII (CD32) subfamily which includes FcγRIIA, FcγRIIB1, FcγRIIB2 and FcγRIIC. B) Amino acid sequence of intracellular domains of FcγRIIA and FcγRIIB2. The brackets indicate the positions of the ITAM of FcγRIIA or the ITIM of FcγRIIB2. The boxes indicate residues crucial to the initiation of endocytosis (different endocytic motifs will be discussed in detail in section 1.4). The yellow arrow points to the FcγRIIB1-specific insertion that prevents coated pit formation and receptor internalization.

FcγRIIA is crucial for allowing signalling from these activating FcγRs in the absence of the Fc receptor γ-chain.

Similar to other activating FcγRs, one important role FcγRIIA plays during the course of immune responses is the removal of IgG-opsonized foreign materials from circulation. FcγRIIA is capable of mediating the engulfment of large IgG opsonized bacteria (>0.5 µm) or particles via phagocytosis. This process requires ITAM-mediated receptor phosphorylation and actin rearrangement (Booth et al., 2002; Indik et al., 1991). FcγRIIA also induces endocytosis of small soluble IgG-containing immune complexes, which is phosphorylation-independent but ubiquitylation-dependent (Mero et al., 2006).

FcγRIIA is a low affinity receptor that interacts only with complexed IgGs (due to avidity effects) but binds monomeric IgG very weakly. It is the only FcγR that binds well to human

IgG2, the main IgG subclass induced in response to bacterial capsular polysaccharides (Takai,

2005; Tan Sardjono et al., 2003). FcγRIIA polymorphism has been associated with higher susceptibility to bacterial infections, which can be attributed to defects in phagocytosis of IgG2- opsonized bacteria (Domingo et al., 2002; Sanders et al., 1995). It has also been linked to the manifestation of autoimmune diseases such as rheumatoid arthritis (Takai, 2005; Tan Sardjono et al., 2003). A major polymorphism in FcγRIIA lies at position 131 where the FcγRIIA gene shows two common allelic variants of which the frequency differs according to ethnicity. The high responder polymorphism R131 has arginine at this position whereas this residue is histidine in the low responder polymorphism H131. These two allotypes differ in their abilities to bind to mouse IgG1 and human IgG2 (Warmerdam et al., 1990; Warmerdam et al., 1991).

FcγRIIA R/R131 genotype is found to associate with higher frequency and earlier onset of SLE symptoms in German SLE patients (Manger et al., 2002). It is thought that this FcγRIIA

polymorphism can constitute a factor that influences the clinical manifestations and the disease course of SLE. However, it does not represent a genetic risk factor for the occurrence of SLE.

FcγRIIB, a family of single chain low-affinity FcγRs, are widely expressed in many cell types in the hematopoietic system. Unlike FcγRIIA, FcγRIIB are present in species other than primates such as mouse, serving as the only inhibitory receptors in the FcγR family. The extracellular domains of FcγRIIA and FcγRIIB share 92 per cent amino acid sequence identity

(Qiu et al., 1990; Su et al., 2007). The divergent residues between the two receptors explain the differences in their binding affinity to IgG subclasses (Syam et al., 2010; Takai, 2005). In contrast, the intracellular domain of FcγRIIB is unrelated to that of FcγRIIA, containing an

ITIM to trigger opposing signalling effects (Fig. 1-3B) (Amigorena et al., 1992; Muta et al.,

1994). The intracellular domains of mouse and human FcγRIIB are largely non-homologous.

A short stretch of sequence containing the ITIM (AENTITYSLL) is conserved between the two.

Two different isoforms of FcγRIIB can be generated through cell type-specific alternative splicing. FcγRIIB1 is present in B cells whereas FcγRIIB2 is mainly expressed in myeloid cells and granulocytes (Hunter et al., 1998; Hunziker and Fumey, 1994). FcγRIIB1 functions mainly as an inhibitory regulator of the action of activating ITAM-containing B cell receptors

(BCR) in B cells. Reconstitution experiments in IIA1.6 B cells have shown that FcγRIIB2 possesses an inhibitory effect similar to FcγRIIB1 (Van den Herik-Oudijk IE et al., 1994).

Both isoforms lack phagocytosis-inducing ability. However, human FcγRIIB2 is able to induce endocytosis of soluble immune complexes (Van den Herik-Oudijk IE et al., 1994). For mouse

FcγRIIB2, a di-leucine motif lying within the ITIM has been shown to be important for endocytosis (Fig. 1-3B) (Hunziker et al., 1994; Matter et al., 1994). This difference in endocytic capacity is due to an FcγRIIB1-specific insertion in the membrane-proximal region

of the cytoplasmic domain, 47 amino acids in the mouse receptor and 19 amino acids in the human receptor, that actively prevents coated pit localization (Miettinen et al., 1992).

Because FcγRIIB plays key roles in modulating pro-inflammatory signals sent by activating receptors, dysfunction of this negative regulator results in an imbalance in the overall immune response and thus the exacerbation of autoimmune diseases (Bolland and Ravetch,

2000; Nimmerjahn et al., 2006; Takai et al., 1996). FcγRIIB is important in the maintenance of peripheral tolerance and the suppression of autoreactive B cell activation. The deletion of inhibitory FcγRIIB in mice leads to induced or spontaneous development of a range of autoimmune phenotypes including elevated Ig levels in response to antigen stimulation, enhanced antibody-induced local and systemic anaphylaxis and attenuated effect of intravenous immunoglobulin (IVIG) treatment (Takai, 2005). Consistent with this, the activated germinal center B cells of a number of mouse strains prone to autoimmune diseases also exhibit reduced levels of FcγRIIB, which has been associated with polymorphisms in the promoter region of the

FcγRIIB gene (Jiang et al., 2000; Jiang et al., 1999; Nimmerjahn et al., 2006; Pritchard et al.,

2000; Xiu et al., 2002). An FcγRIIB polymorphism encoding a single amino acid substitution from isoleucine to threonine at position 232 (I232T) in its transmembrane domain has been found at a higher ratio in Asian but not Caucasian SLE patients (Chu et al., 2004; Kyogoku et al., 2002; Li et al., 2003; Siriboonrit et al., 2003). Further studies suggest that the I232T polymorphism causes the exclusion of FcγRIIB from lipid rafts, leading to the inability of the receptor to inhibit activating receptors. The balanced positive and negative signalling during an inflammatory response is disrupted resulting in the promotion of SLE (Floto et al., 2005).

Both FcγRIIA and FcγRIIB2 are widely expressed on human myeloid cells, but their expression levels and relative ratio vary depending on the cell types, external stimuli including changes in the cytokine milieu and the state of cell maturation. Several studies have been

carried out to assess their expressions both at the mRNA transcript level and at the protein level.

FcγRIIA was found to be expressed at comparable levels in monocytes, macrophages, immature DCs (iDCs) and mature DCs (mDCs) (Guriec et al., 2006; Liu et al., 2006; Su et al.,

2007). In contrast, the levels of both FcγRIIB1 and FcγRIIB2 are reduced upon DC maturation, reflecting differential roles of iDCs and mDCs during immune responses (Guriec et al., 2006;

Liu et al., 2006). Although both isoforms of FcγRIIB are present in monocytes and mDCs,

FcγRIIB2 is preferentially expressed (Su et al., 2007; Tridandapani et al., 2002).

Table 1-1 Summary of research findings on the expressions of FcγRIIA and FcγRIIB and the effects of cytokine regulation in primary human myeloid cells

cell type FcγRIIA and FcγRIIB level cytokine treatment Treatment with IL-4a or IL-4+IL- g Much more FcγRIIA than FcγRIIBd. 10 leads to increase in FcγRIIB, but monocytes has little effect on FcγRIIAg. a More FcγRIIB2 than FcγRIIB1 . IL-10 treatment increase both FcγRIIA and FcγRIIB levelsf. express high amounts of FcγRIIA and FcγRI macrophages -- but very little FcγRIIBb express both FcγRIIA and FcγRIIB (more iDC FcγRIIB than FcγRIIA) but very little -- FcγRIb Maturation leads to decrease in FcγRIIB1 and FcγRIIB2c. mDCs have low, heterogeneous (between IL-10 treatment leads to increase in mDC and within donors, varied several-fold the ratio of FcγRIIA to FcγRIIBb among donors) FcγRIIB expressiond. IL-10+IL-13 decrease the ratiob More FcγRIIB2 expression than FcγRIIB1d. IL-10 treatment increases both Express both FcγRIIA and FcγRIIB FcγRIIA and FcγRIIB levelse moDC (although hetergeneous) and very little IFNγ increases FcγRIIA and FcγRIe. decreases FcγRIIB2e a: (Tridandapani et al., 2002): total protein level by western blotting, surface protein level by flow cytometry, mRNA level by RT-PCR b: (Liu et al., 2006): total protein level by western blotting, surface protein level by flow cytometry, mRNA level by RT-PCR c: (Guriec et al., 2006): total protein level by western blotting, surface protein level by flow cytometry, mRNA level by RT-PCR d: (Su et al., 2007): surface protein level by flow cytometry, mRNA level by RT-PCR e: (Boruchov et al., 2005): surface protein level by flow cytometry f: (Liu et al., 2005): surface protein level by flow cytometry, mRNA level by RT-PCR g: (Wijngaarden et al., 2004): surface protein level by flow cytometry,

The ratio of expression of FcγRIIA to FcγRIIB can be regulated by cytokines. IL-4, a

Th2 cytokine, in combination with IL-10 can highly upregulate the expression of FcγRIIB on human monocytes whereas the FcγRIIB level is decreased by the Th1 cytokine IFNγ (Joshi et al., 2006; Pricop et al., 2001; Tridandapani et al., 2002). The protein ratio of FcγRIIA over

FcγRIIB on mDCs increases with the treatment of IFNγ or IL-10 and decreases with the combined treatment of IL-13 plus IL-10 or IL-4 plus IL-13 (Boruchov et al., 2005; Guriec et al.,

2006; Liu et al., 2006; Liu et al., 2005). These findings reveal an intricate modulation of the overall expression of activating and inhibitory FcγRs to ensure effective yet not prolonged immune responses.

1.3 Binding affinity of FcγRs to IgG subclasses

Besides the multiplicity of the FcγR family, the existence of four subclasses of IgG in human (IgG1-IgG4) and mouse (IgG1, IgG2a, IgG2b and IgG3) adds another layer of complexity as each isotype binds to different FcγRs with varying affinity and specificity. FcγR specificities for IgG subclasses in the mouse system have been extensively studied. Mouse

IgG2a and IgG2b are more potent than mouse IgG1 and IgG3 in triggering antibody-mediated host responses (Nimmerjahn et al., 2008). This is because the binding affinity of mouse inhibitory receptor FcγRIIB, to mouse IgG1 is 10 fold higher than FcγRIII, the main mouse activating receptor that binds to mouse IgG1 (Nimmerjahn et al., 2005). In contrast, IgG2a and

IgG2b, which are functionally dependent on mouse activating receptor FcγRIV, bind to FcγRIV with affinities 70- or 7-fold higher than to the inhibitory receptor. These findings suggest that

IgG1 activity is more tightly regulated by changes in the expression level of FcγRIIB than

IgG2a and IgG2b.

In the human system, the four IgG isotypes are differentially generated in response to different antigens (Bruhns et al., 2009). IgG1 and IgG3 are elicited by T cell-dependent protein-derived antigens whereas T cell-independent carbohydrates such as bacterial polysaccharides elicit the production of IgG2. Unlike the situation in the mouse system, the inhibitory receptor FcγRIIB exhibits much lower affinities to all IgG isotypes except for IgG4 than the activating receptors including FcγRIIA, the main receptor for IgG2. This discrepancy between human and mouse systems in the binding affinities of activating and inhibitory FcγRs to IgG subclasses may have significant implications in the regulation of proinflammatory effector functions in response to different stimuli as well as the processing and presentation of internalized antigens. FcγRIIA is preferentially recruited over FcγRIIB2 to the site of particle binding during phagocytosis of antibody-opsonized particles (Syam et al., 2010). This local enrichment of the activating receptor relative to its inhibitory counterpart is mainly due to the differences at two key residues in the antibody binding site between the extracellular domains of the two receptors which result in higher binding affinity of FcγRIIA to IgG1.

Table 1-2 Binding of IgG isotypes to FcγRs

human (Bruhns et al., 2009) receptor IgG binding preference (Ka) IgG FcγR binding preference (Ka) 107M‐1 IgG1=IgG3>IgG4. 107M‐1 FcγRI>FcγRIIA>FcγRIIIA>>FcγRIIB/C, FcγRI IgG1 no IgG2 binding FcγRIIIB 105M‐1 FcγRIIA>>FcγRIIIA,FcγRIIB/C. FcγRIIA 106 M‐1 IgG1>IgG3>IgG2>IgG4 IgG2 FcγRI and FcγRIIIB do not bind

107M‐1 FcγRI>FcγRIIIA>FcγRIIA, FcγRIIB/C 105M‐1 IgG4>IgG3>IgG1>IgG2 IgG3 FcγRIIIB>>FcγRIIB/C 107M‐1 FcγRI>>FcγRIIIA,FcγRIIA,FcγRIIB/C. FcγRIIIA 106M‐1 IgG3>IgG1>IgG2>IgG4 IgG4 FcγRIIIB do not bind 106M‐1 IgG3>IgG1. FcγRIIIB no IgG2 or IgG4 binding mouse (Nimmerjahn et al., 2005; Saylor et al., 2010) 109M‐1 IgG2a>>IgG3. 106M‐1 FcγRIIB>FcγRIII. FcγRI and FcγRIV do FcγRI IgG1 no IgG1 or IgG2b binding not bind 106M‐1 IgG1=IgG2b>IgG2b. FcγRIIB IgG2a 109M‐1 FcγRI>FcγRIV>>FcγRIIB=FcγRIII no IgG3 binding 105M‐1 IgG2a=IgG2b=IgG1. 107M‐1 FcγRIV>FcγRIIB>FcγRIII. FcγRI do FcγRIII IgG2b no IgG3 binding not bind 107M‐1 IgG2a>IgG2b. FcγRIV IgG3 only FcγRI binds to IgG3 weakly. no IgG1 or IgG3 binding

1.4 Endocytosis

Endocytosis of soluble molecules from the cell surface can be categorized into two classes: clathrin-dependent endocytosis and clathrin-independent processes (Aguilar and

Wendland, 2005). The clathrin-independent endocytosis pathways that generally depend on cholesterol-rich membrane domains are less understood (Kirkham and Parton, 2005). These processes include caveolae-dependent endocytosis, non-caveolar clathrin-independent pathways and macropinocytosis. The best characterized endocytic process is clathrin-dependent receptor- mediated endocytosis. In this case, clathrin is recruited to ligand-engaged receptors on the cell surface with the help of one of several adaptor proteins such as clathrin-associated adaptor protein 2 (AP2) (Maxfield and McGraw, 2004; Motley et al., 2003; Traub, 2003). Clathrin- coated pits are assembled by forming spherical cages with assistance from accessory proteins 15

before budding off. Some examples of receptors that undergo clathrin-dependent endocytosis are transferrin receptor and FcγRIIB2 (Iacopetta et al., 1988; Mousavi et al., 2007). FcγRIIA- mediated endocytosis is clathrin-dependent but AP2-independent (Mero et al., 2006).

After the removal of the clathrin coat, the newly formed vesicles fuse with endosomes and mature into early/sorting endosomes (Fig. 1-4). This is where the first major intracellular sorting event of receptors and their ligands occurs. The lumenal pH of early/sorting endosomes is about 6.0 (Maxfield et al., 2004). This low pH favours the release of ligands from their receptors. At this stage, receptors are targeted to different trafficking routes by geometry-based sorting. Receptors destined for recycling are concentrated in narrow-diameter tubules with surface-area-to-volume ratio higher than the vesicle portion of early/sorting endosomes (Dunn et al., 1989; Maxfield et al., 2004; Mayor et al., 1993). As these tubules are pinched off, the recycling membranes containing receptors are preferentially sorted away from soluble molecules in the early/sorting endosomes. These receptors are transported rapidly to recycling endosomes before being recycled back to cell surface. It is thought that membrane receptors without any specific targeting signal are generally sorted for recycling by this geometry-based sorting mechanism (Maxfield et al., 2004). Transferrin receptor and low-density- lipoprotein

(LDL) receptor are well characterized transmembrane proteins known to go through the recycling sorting route (Presley et al., 1997; van Kerkhof et al., 2005). However, unlike LDL that is degraded in lysosomes, transferrin remains bound to its receptor in the acidic environment of early/sorting endosomes before being transferred back to the cell surface.

In contrast, specific targeting signals are required to deliver receptors to late endosomes for degradation (lysosomal sorting motifs will be discussed in detail later in this section).

These receptors are sorted into late endosomes as endosomal maturation occurs (Fig. 1-4). The lumenal pH of the endosomes drops progressively to 5.0-6.0, a process called endosomal

Fig. 1-4 Endosomal compartments and the trafficking of cell surface receptors (adapted from Maxfield FR, 2004) Internalization and intracellular sorting of four transmembrane receptors through various endosomal compartments. The transferrin receptor binds to transferrin; the low-density lipoprotein receptor (LDLR) binds to low-density lipoprotein (LDL); the cation-independent mannose-6-phosphate receptor (CI-MPR) binds to lysosomal enzymes; epidermal growth factor receptor (EGFR) binds to epidermal growth factor. These receptors are concentrated in clathrin- coated pits after ligand binding followed by recruitment of clathrin, AP2 and accessory proteins. The clathrin-coated pit pinches off. Following the removal of the clathrin coat, these receptors along with their ligands are delivered to early/sorting endosomes. At this stage, some receptors such as transferrin receptor bound to transferrin and LDLR are transported to recycling endosomes by geometry-based sorting before trafficking back to cell surface. Other receptors such as EGFR and CI-MPR are retained in early/sorting endosomes as endosomal maturation occurs. In late endosomes/multivesicular body (MVB), EGFR along with its ligand are transported into the inner vesicles of MVB and eventually get degraded in lysosomes. Some of the internalized ligand-bound CI-MPR are sorted to recycling endosomes through early sorting endosomes. Some CI-MPR access the trans-Golgi network from late endosomes after lysosomal enzymes dissociate from CI-MPR. These free CI-MPR are delivered to recycling endosomes before recycling back to the cell surface. 17

maturation. This acidic environment allows for the dissociation of ligands from receptors. A second sorting event can occur in late endosomes where receptors removed from late endosomes access the trans-Golgi network through small vesicles and then recycle back to cell surface after reaching recycling endosomes. Some examples of receptors that undergo such recycling route through late endosomes are DC receptor DEC-205 (Mahnke et al., 2000) and cation-independent mannose-6-phosphate receptor (CI-MPR) (Ghosh et al., 2003). Receptors and ligands remaining in late endosomes eventually get degraded as late endosomes fuse with lysosomes. The lumenal pH continues decreasing and lysosomal proteases become activated.

The degradation kinetics of different molecules vary, reflecting their intrinsic susceptibilities to lysosomal proteolysis (Trombetta et al., 2003). Many signalling receptors are targeted for lysosomal degradation which serves as a mechanism for signal termination. Examples of receptors that are targeted for degradation include BCR and epidermal growth factor receptor

EGFR (Dikic, 2003; Gondre-Lewis et al., 2001).

Early/sorting endosomes and recycling endosomes are relatively smaller in size with diameters of about 60nm (Hopkins, 1983; Maxfield et al., 2004; Yamashiro et al., 1984). They are tubular organelles associated with microtubules, located towards the cell periphery

(Katzmann et al., 2002). In contrast, late endosomes are larger (about 300–400 nm in diameter) and more spherical compared to early/sorting endosomes and recycling endosomes (Gruenberg,

2001). They are located more towards the centre of cells, close to the nucleus. A subset of late endosomes with multivesicular morphology, termed multivesicular bodies (MVB), is of particular interest. These organelles possess a special sorting mechanism for the separation of molecules to be degraded from those that are recycled to cell surface. The limiting membrane of MVBs pinches inward and receptors with degradation sorting signals are delivered to lumenal vesicles. One of the signals that regulate this process is ubiquitylation at the

cytoplasmic domain of receptors. It is known that EGFR on the limiting membrane of MVB is delivered to the lumenal vesicles with the help from ESCRT complexes upon receptor ubiquitylation (Longva et al., 2002)

A variety of protein markers are commonly used for experimental identification of various endosomal compartments. Different members of the Rab family, GTPase regulatory proteins with important roles in membrane transport, are associated with different parts of the endosomal system. Rab5 and early endosome antigen 1 (EEA1), two markers for early/sorting endosomes, are involved in regulating fusion of newly formed vesicles with early/sorting endosomes. Another member of the Rab family, Rab7, is important for the biogenesis and maintenance of lysosomal compartments (Bucci et al., 2000). Rab7 and lysosomal-associated membrane protein 1 (Lamp1) are often used as markers to study receptor localization in late endosomes/lysosomes. The co-localization of internalized molecules with Rab11, involved in recycling back to the cell surface, marks the sorting of these molecules to recycling endosomes.

The trafficking of transmembrane proteins to specific endosomal compartments can be mediated by distinct sorting signals. Present in the cytoplasmic domain of receptors, these signals generally consist of short stretches of amino acid residues. Besides driving the internalization of cell surface receptors, many of these motifs are also lysosomal-targeting signals. The tyrosine-based motif NPXY (X can be any amino acid) mediates rapid internalization of numerous receptors including members of the LDL receptor family, EGFR and insulin receptor (Bonifacino and Traub, 2003). NPXY recognizes both the clathrin terminal domain and the μ2 subunit of AP-2 to mediate clathrin-dependent AP2-dependent endocytosis. It has also been shown to be involved in targeting LDL receptor-related protein for recycling through interaction with sorting nexins (van Kerkhof et al., 2005). Another tyrosine-based motif is YXXΦ where Φ stands for an amino acid residue with a bulky

hydrophobic side chain. This motif can be found in many transmembrane receptors including endocytic receptors such as transferrin receptor, lysosomal membrane proteins such as LAMP1, trans-Golgi network proteins such as furin and the major histocompatibility complex class-II

(MHC class-II) molecule HLA-DM. The YXXΦ motif not only acts as an endocytic signal for rapid internalization of these proteins, it is also important for the sorting of these proteins into lysosomal compartments. Its interaction with AP complexes, especially AP-2, has been extensively studied. There are three tyrosine residues in the intracellular domain of FcγRIIA with two in the ITAM motif (Fig. 1-3B). Whether they may serve as potential YXXΦ targeting signals is unknown.

Like these two tyrosine-based targeting signals, di-leucine based motifs [DE]XXXL[LI] and DXXLL have been implied in mediating both endocytosis and lysosomal transport of transmembrane proteins (Bonifacino et al., 2003). In the [DE]XXXL[LI] signal commonly found in transmembrane proteins such as CD3γ and CD4, the first leucine is invariant whereas the second can be a leucine or an isoleucine. The acidic residue four amino acids upstream of the first leucine has been reported to be important for lysosomal targeting but not for endocytosis. Similar to tyrosine-based motifs, the [DE]XXXL[LI] motif also interacts with AP complexes. It was shown by alanine scanning that the di-leucine motif in the intracellular domain of mouse FcγRIIB2 is crucial for triggering its endocytosis (Hunziker et al., 1994).

The cytoplasmic tails of human and mouse FcγRIIB2 are highly conserved only in a short amino acid stretch that includes the ITIM and the di-leucine motif (ENTITYSLL). While there is no direct evidence, it is likely the di-leucine motif also serves as an endocytic signal in the human receptor.

1.5 Intracellular trafficking and antigen presentation of FcγR

Efforts have been made to analyze the intracellular sorting of FcγRs into different endocytic compartments after engagement with multivalent immune complexes. As early as the 1980s, before the identification of various FcγR subclasses, studies in mouse macrophages showed that the degree of receptor clustering determines its intracellular fate (Mellman et al.,

1984; Ukkonen et al., 1986). While monovalent ligands bound to FcγRs were rapidly recycled back to cell surface, receptors were rerouted away from the recycling pathway and delivered to lysosomes for degradation upon stimulation with polyvalent immune complexes (Mellman et al., 1984; Mellman et al., 1984).

Among the members of the FcγR family, the intracellular trafficking of the activating

FcγRI has been well characterized. Using human monocytic cell lines or transfected cell models, it was found that FcγRI is diverted from recycling and targeted for lysosomal degradation when cross-linked (Harrison et al., 1994). This sorting event is dependent on γ- chain-mediated recruitment and activation of tyrosine kinases such as Syk, as well as PI3K

(Bonnerot et al., 1998; Gillooly et al., 1999; Norman et al., 1998). A role for γ-chain in directing receptors for lysosomal degradation has also been observed in FcαR, the Fc receptor for IgA (Launay et al., 1999). One form of FcαR containing two γ-chains was found in endo- lysosomal compartments whereas the other form unassociated with γ-chains accessed the recycling pathway. A recent study in a transfected cell model indicates that internalized FcεRI is also routed towards lysosomes for degradation after engagement for endocytosis (Fattakhova et al., 2009). Downregulation of activating FcγRIIA has been demonstrated in human neutrophils where FcγRIIA is rapidly internalized and degraded within two minutes after cross- linking (Barabe et al., 2002; Marois et al., 2009). This rapid degradation of FcγRIIA is SFK- dependent (Barabe et al., 2002). FcγRIIA also plays a role in the pathogenesis of SLE during

which FcγRIIA delivers endogenous DNA-containing autoantibody complexes to lysosomes containing toll-like receptor 9 (TLR9), leading to the activation of plasmacytoid DCs (Means et al., 2005).

Unlike the degradation of ligands taken up by activating receptors, evidence suggests that immune complexes internalized by FcγRIIB2 on mouse DCs can access a nondegradative pathway, allowing for recycling of antigen to plasma membrane for T-cell independent B cell priming (Bergtold et al., 2005). The recycling of FcγRIIB2 with or without immune complex stimulation has also been shown directly in rat liver endothelial cells (Mousavi et al., 2007).

However, ligands bound to mouse FcγRIIB2 expressed heterologously in FcγR-negative fibroblasts are internalized and delivered to lysosomes rather than cell surface (Miettinen et al.,

1989).

Besides the important role FcγRs play in the removal of immune complexes from circulation and induction of inflammatory responses, FcγR-mediated endocytosis of immune complexes can also enhance the efficiency of presentation of antigen-derived peptides on the surface of APCs to activate both CD4+ and CD8+ T cells by three to four orders of magnitude compared to presentation of antigen taken up in fluid phase (Amigorena and Bonnerot, 1999).

It has been shown that, besides containing lysosomal targeting signal, the intracellular domain of human FcγRI-α chain can promote MHC class-II-mediated antigen presentation, independently of a functional Fc receptor γ-chain ITAM (van Vugt et al., 1999). Studies carried out in transfected cell models show that different endocytosis-competent FcγRs, namely mouse

FcγRIII and FcγRIIB2, can increase presentation of antigen-derived peptides via MHC class-II molecules. However, they differ in their ability to present different T cell epitopes from the same antigen (Amigorena et al., 1998; Regnault et al., 1999). This implies important differences in processing after internalization.

Most of the processing and loading of internalized exogenous antigen onto MHC class-II occurs in MHC class-II containing compartments (MIIC), a subset of late endosomes often multivesicular in nature and rich in MHC class-II molecules (Stern et al., 2006). Notably, for

BCR, binding of antigen to BCR results in the reorganization, fusion and acidification of endosomal vesicles into an MHC class-II rich complex of large vesicles and the targeting of antigen-BCR complexes to these late endosomes (Siemasko et al., 1998). The delivery of antigen-BCR complexes to MIIC and the presentation of antigen-derived peptides requires ubiquitylation (Drake et al., 2006)

In addition, in DCs, exogenous antigen internalized in immune complexes via FcγRs can also be cross-presented onto MHC class-I molecules to activate CD8+ T cells as antigen enters the cytosol from endososomal/phagosomal compartments (Rodriguez et al., 1999). This is followed by proteasomal degradation of the antigen and transfer of antigen-derived peptides to the endoplasmic retinulum via TAP where they are loaded onto MHC class-I. However, the detailed mechanism of where and how this translocation of internalized antigen into the cytoplasm occurs is unclear (Ackerman and Cresswell, 2004; Ackerman et al., 2006; Burgdorf et al., 2007; Villadangos et al., 2007).

1.6 Receptor ubiquitylation and phosphorylation in FcγR-mediated endocytosis

Ubiquitylation, also known as ubiquitination or ubiquitinylation, is a posttranslational modification process by which ubiquitin, a 76 amino acid globular protein that is highly conserved in eukaryotes, is covalently conjugated to target proteins (Staub and Rotin, 2006).

Ubiquitin is usually conjugated to lysine residues of the target proteins or other ubiquitin moieties, although it has been demonstrated that some other residues such as cysteine and

serines can also function as ubiquitylation sites when lysines are not present (Cadwell and

Coscoy, 2005; Wang et al., 2007).

The ubiquitylation process is facilitated by several enzymes in a multistep fashion. Three classes of enzymes are involved, including 1) E1: ubiquitin-activating enzyme; 2) E2: ubiquitin-conjugating or ubiquitin-carrier enzyme; 3) E3: ubiquitin-protein ligase (Fig. 1-5). E1 first activates free ubiquitin in an ATP-dependent manner by forming a thioester bond between its catalytic cysteine and the carboxyl terminus of ubiquitin (Staub et al., 2006; Weissman,

2001). The ubiquitin moiety is then transferred to one of the many E2 enzymes through a thioester linkage. After the association of E2 to an E3 enzyme and recruitment of a target protein, E3 facilitates the conjugation of ubiquitin to the target protein. E4 enzymes along with

E3 ligases are important for the formation of the polyubiquitin chain whereas deubiquitinating enzymes (DUBs) reverse the ubiquitylation process.

Ubiquitylation plays important roles in the regulation of many cellular processes such as proteasome-mediated protein degradation, membrane protein transport and processing, cell cycle progression, DNA repair, cell proliferation and differentiation, apoptosis and stress responses (Weissman, 2001). To maintain a tight control over these processes, precise targeting of each ubiquitylated protein to its designated route must be ensured. There are two ways the specificity is generated. First of all, although only about ten E1 exist in humans, there are more than a hundred E2 and about a thousand E3 (Hicke et al., 2005; Staub et al., 2006).

This large number of enzymes allows for specific recognition of target proteins during ubiquitylation.

Furthermore, ubiquitins can be linked to proteins in different ways, which alter the fate of the proteins. Different ubiquitin linkages can be formed, serving as distinct signals for various cellular processes. It is commonly believed that polyubiquitylation (several ubiquitin

Fig. 1-5 Ubiquitylation process of target protein Ubiquitin is activated by a ubiquitin-activating enzyme, E1, in an ATP-dependent manner and interact with the active site of E1 through thioester bond formation. This is followed by transfer of ubiquitin to the active site of a ubiquitin-conjugating enzyme, E2. With the assistance of E3 ubiquitin ligases, ubiquitin is transferred to the target protein either directly from the E2 or through E3 as an intermediate. E4 enzymes promote the formation of polyubiquitin chains whereas deubiquitylating enzymes disassemble the polyubiquitin chains.

molecules on one lysine) targets cytosolic proteins for degradation by the 26S proteasome, one of the well-known functions of ubiquitylation (Pickart, 2000). In contrast, monoubiquitylation

(one ubiquitin on one lysine) or di-ubiquitylation (two ubiquitins on one lysine) are found in membrane protein endocytosis and transport (Staub et al., 2006). However, evidence suggests that it is actually polyubiquitylation or multiubiquitylation (monoubiquitylation on several lysines) instead of a single ubiquitin moiety that acts as an internalization signals to drive efficient endocytosis (Barriere et al., 2006).

Besides the length of the polyubiquitin chain, the choice of lysines on ubiquitin when building the polyubiquitin chain also determines the fate of the target protein. While polyubiquitylation linked through lysine 48 (K48) of ubiquitin promotes proteasomal protein degradation, lysine 63 (K63) linkages are important in a range of functions (Weissman, 2001;

Xu et al., 2009). One of these is to signal membrane protein endocytosis and sorting either for lysosomal degradation or receptor recycling (Haglund et al., 2003; Hicke, 2001; Katzmann et al., 2002; Shih et al., 2000). During EGFR-mediated endocytosis, EGFR is polyubiquitylated and multiubiquitylated upon ligand binding (Huang et al., 2006a). A majority of the polyubiquitin chains are linked through K63.

Studies in both yeast and mammalian systems have indicated an important role of ubiquitylation in the regulation of trafficking of many membrane proteins (Shin et al., 2006;

Staub et al., 2006). Ubiquitylation signals a receptor to undergo endocytosis through interaction with the ubiquitin binding domains of endocytic adaptor proteins (Hurley et al.,

2006; Madshus and Stang, 2009). The initiation of growth hormone receptor (GHR) endocytosis requires the participation of the ubiquitin-conjugation system (Strous et al., 1996).

Ubiquitylation has also been implicated in the internalization of EGFR (Goh et al., 2010;

Huang et al., 2007; Sigismund et al., 2005; Stang et al., 2004). Ubiquitylation of FcγRIIA

occurs prior to the assembly of clathrin-coated pits in a manner independent of receptor phosphorylation by SFK (Mero et al., 2006). As phosphorylation of receptors is usually considered to be required to trigger receptor ubiquitylation, these findings point to a novel mechanism of FcγR endocytosis.

Besides the initial step of ligand internalization at the cell surface, ubiquitylation is also involved in the subsequent intracellular sorting of receptors into specific endosomal compartments for their downregulation (Katzmann et al., 2002; Staub et al., 2006). For example, EGFR polyubiquitylation by E3 ligase c-Casitas B-lineage lymphoma (c-Cbl) is sustained after internalization (Umebayashi et al., 2008). Receptor ubiquitylation is essential for EGFR sorting for lysosomal degradation (Duan et al., 2003; Grovdal et al., 2004; Huang et al., 2006a; Ravid et al., 2004). A key step of this sorting event occurs in multivesicular bodies

(MVB), the transport intermediates between early and late endosomes (Longva et al., 2002).

Hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs), which contains a ubiquitin- interacting motif (UIM), targets the ubiquitylated receptor and its cargo to the limiting membrane of MVBs. (Grovdal et al., 2004; Huang et al., 2006a; Hurley and Emr, 2006; Ravid et al., 2004) (Fig. 1-6). Hrs then delivers cargo to endosomal sorting complex required for transport-I (ESCRT-I) complexes by interacting with tumor susceptibility gene 101 (Tsg101) followed by transfer of cargo to the inner vesicles of MVB by ESCRT-II and ESCRT-III complexes by inward budding. The limiting membrane then fuses with lysosomes, resulting in the degradation of inner vesicles along with their contents. Deubiquitylation also appears to have an important regulatory role at this step. Two ubiquitin isopeptidases, associated molecule with the SH3-domain of STAM (AMSH) and ubiquitin-specific processing protease Y (UBPY), de-ubiquitylate EGFR with opposite effects on EGF degradation: AMSH negatively regulates

EGFR sorting to lysosomes whereas UBPY is essential for EGFR degradation (Alwan and van

Leeuwen, 2007; McCullough et al., 2004; Row et al., 2006).

Another example of ubiquitylation playing an essential role in membrane receptor trafficking is that of GHR. It was found that the ubiquitin conjugating system is required for

GHR-mediated endocytosis and receptor degradation in lysosomal compartments upon growth hormone engagement (Strous et al., 1996). Conversely, GHR ubiquitylation in turn depends on an intact endocytic pathway, suggesting a cooperation between the two systems to modulate

GHR internalization (Govers et al., 1997). GHR itself can be ubiquitylated on multiple lysine residues in its cytoplasmic tail with polyubiquitin chains formed at each site. However, receptor ubiquitylation per se is not essential for the ligand-induced GHR endocytosis (Govers et al., 1999). Instead, the binding of a ubiquitin ligase SCF (βTrCP) to a unique ubiquitin- dependent endocytosis (UbE) motif in the intracellular domain of GHR, but not GHR ubiquitylation, is important in regulating its endocytosis and degradation (Govers et al., 1999; van Kerkhof et al., 2007). It exerts its action through controlling the incorporation of ligand- bound GHR into clathrin-coated vesicles (Sachse et al., 2001).

Another important player in the sorting of receptors upon ligand-induced endocytosis is the activity of the proteasome, although the target of proteasome action is unknown (Longva et al., 2002; Strous and van Kerkhof, 2002). The ubiquitin-proteasome pathway is essential for the endosomal sorting of selected membrane proteins for lysosomal degradation, but not soluble proteins that dissociate from their receptors (van Kerkhof et al., 2001). These findings indicate an additional indirect role of the proteasome in regulating lysosomal degradation of endocytosed proteins. Alternatively, this can be a result of depleting free ubiquitins available for receptor ubiquitylation by inhibition of proteasome activity.

Besides EGFR and GHR, ubiquitylation also plays a key role in the regulation of ligand-

Fig. 1-6 The ESCRT complexes in MVB sorting of EGFR (adapted from Hurley and Emr, 2006) Hrs transfers ubiquitylated EGFR-EGF complexes to the limiting membrane of MVB vesicles by binding to PI3P via its FYVE domain and to ubiquitin on EGFR via its ubiquitin-interacting motif (UIM). ESCRT-I complex including Tsg101 recognizes ubiquitylated EGFR via interacting with Hrs followed by activation of ESCRT-II. ESCRT-III concentrates EGFR into lumenal MVB vesicles. Enzymes AMSH and UBPY de-ubiquitylate EGFR before EGFR enters MVB vesicles.

induced endocytosis and the degradation of membrane receptors in the hematopoietic system, such as BCR, T cell receptor (TCR) and MHC class-II. The intracellular trafficking of antigen-

BCR complexes into MHC class-II-containing MVB and subsequent antigen presentation depend on BCR ubiquitylation (Drake et al., 2006). Down-regulation of MHC class-II from the cell surface of iDCs by endocytosis is mediated by the ubiquitylation of MHC class-II β-chain cytoplasmic tail (Shin et al., 2006). Genetic knock-out studies provide strong evidence that

TCR ubiquitylation upon engagement with peptide-MHC negatively regulates its signalling and stability through promoting receptor endocytosis and sorting for lysosomal degradation (Staub et al., 2006).

Engagement of FcεRI on mast cells and basophils results in Syk-dependent receptor ubiquitylation at multiple lysine residues in the intracellular domains of FcεRI β and γ subunits by E3 ligase c-Cbl (Molfetta et al., 2009; Paolini et al., 2002). Syk and Cbl mutually regulate each other’s activities. Receptor ubiquitylation is essential for the induction of FcεRI-mediated endocytosis and partially required for Hrs-mediated intracellular sorting of FcεRI for lysosomal degradation (Molfetta et al., 2009).

It has been found that different signals are required for FcγRIIA-mediated endocytosis of soluble immune complexes and phagocytosis of large antibody opsonised particles (Booth et al.,

2002). The endocytosis of FcγRIIA depends on receptor ubiquitylation whereas ITAM- mediated receptor phosphorylation is essential for inducing its phagocytosis. Ubiquitylation of

FcγRIIA occurs at the plasma membrane before its endocytosis (Mero et al., 2006). These two processes are closely linked and the inhibition of proteasome action abolishes both activities.

FcγRIIA ubiquitylation includes the formation of polyubiquitin chains, which does not occur obligatorily through either K63 or K48 linkage (Mero et al., 2006). During phagosomal maturation, FcγRs are removed from phagosomes to punctate acidic endosomal MVBs through

vesicle budding (fission). Ubiquitylation is required for the formation of these multivesicular structures (Lee et al., 2005).

Receptor phosphorylation and the activation of protein kinases such as SFK, Syk and

PI3K in FcγR-mediated signalling and phagocytosis have been extensively characterized (Cox et al., 1999; Fitzer-Attas et al., 2000; Kiefer et al., 1998; Kim et al., 2001; Kwiatkowska et al.,

2003; Majeed et al., 2001). However, their roles in endocytosis of immune complexes by

FcγRs remain less well understood. There has been controversy over the role of the protein kinase Syk in FcγR-mediated endocytosis of immune complexes. By overexpression of dominant negative mutant Syk and introduction of point mutations in the ITAM of FcγR-γ chain that affect Syk activation, it was shown that Syk is not necessary for the internalization of immune complexes, but important for cell signalling and for the delivery of receptors to lysosomes (Bonnerot et al., 1998). Studies in cells transfected with FcγRI and in human monocytes also suggest that the recruitment and activation of Syk upon receptor clustering with immune complexes are not required for the initiation of its endocytosis (Norman et al., 1998).

In contrast, Syk is important for FcγR-mediated immune complex internalization and subsequent antigen presentation to T cells in mouse bone marrow-derived DCs (Sedlik et al.,

2003). In transfected cell lines, mouse macrophages and human monocytes, it was found that, in contrast to the phagocytosis pathway, FcγR-mediated endocytosis is independent of receptor phosphorylation, Syk, SFK and PI3K (Huang et al., 2006b; Mero et al., 2006).

1.7 Project aims and thesis outline

The opposite signalling functions of activating and inhibitory FcγRs have been extensively studied, but their trafficking after internalization is less clear. A number of studies have characterized to some extent the intracellular sorting of Fcγ receptors in rodents. Early

studies in the 1980s showed that the clustering of FcγRs on mouse macrophages with immune complexes results in lysosomal degradation of both ligands and receptors (Mellman et al., 1984;

Ukkonen et al., 1986). However, these studies were carried out before the identification of various low affinity FcγRs. In addition, the anti-FcγR antibody 2.4G2 used in their experiments does not differentiate mouse FcγRIIB2 and FcγRIII. Utilizing mice deficient in either FcγR-γ chain or FcγRIIB2, Bergtold et al demonstrated the divergent sorting routes of immune complexes internalized by activating or inhibitory FcγRs in mouse bone marrow-derived iDCs

(Bergtold et al., 2005). While they investigated the fate of immune complexes internalized via either of the two endocytic pathways, no direct evidence was provided regarding the trafficking behaviours of the FcγRs themselves. Another study showed that, in contrast to FcγRIIB2 in mouse iDCs that recycles intact antigens to cell surface, FcγRIIB2 expressed on transfected cells delivers immune complexes to lysosomes (Miettinen et al., 1989). FcγRIIB2 in rat liver sinusoidal endothelial cells accesses the recycling pathway after internalization of immune complexes (Mousavi et al., 2007). Unlike the receptors, immune complexes internalized by

FcγRIIB2 in these cells are degraded in lysosomes. These differences in ligand fate may be due to the more alkaline endosomal compartments of iDCs with less efficient lysosomal proteolysis.

This leads to limited capacity for antigen degradation after internalization compared to mDCs, macrophages and even fibroblasts (Delamarre et al., 2005; Trombetta et al., 2003).

To date, there have been very few studies on the trafficking behaviours of human FcγRs during ligand-induced endocytosis. The understanding of the endocytosis and subsequent intracellular transport of FcγRIIA has been especially limited. Rapid degradation of FcγRIIA has been observed in human neutrophils (Barabe et al., 2002). This degradation requires both cholesterol-rich domains and SFK. FcγRIIA downregulation in these cells appeared to be mediated through a proteasomal pathway and dependent on receptor ubiquitylation by E3 ligase

c-Cbl (Marois et al., 2009). Dai et al suggests that unlike FcγRI, FcγRIIA does not access

LAMP1-positive lysosomal compartments upon antibody-mediated aggregation (Dai et al.,

2009). However, only 10 minutes were allowed for receptor clustering and internalization which may not be long enough for the lysosomal delivery of FcγRIIA. In addition, visualization of lysosomal compartments and their co-localization with FcγRIIA was unclear.

The intracellular sorting of both human FcγRIIA and FcγRIIB2 which has not yet been investigated is addressed in Chapter 3.

We have gained some understanding of the molecular mechanism that underlies the endocytosis pathway mediated by human FcγRIIA such as its requirement for ubiquitylation independent of receptor phosphorylation and the involvement of clathrin but not AP2 (Booth et al., 2002; Mero et al., 2006). The endocytosis of mouse FcγRIIB2 has been found to be driven by a di-leucine motif (Hunziker et al., 1994; Matter et al., 1994). Not much is known about the internalization of human FcγRIIB2 other than its capability to take up soluble immune complexes (Van den Herik-Oudijk IE et al., 1994). Another question that remains elusive is whether there are any differences in the rate of immune complex uptake by FcγRIIA versus

FcγRIIB2. These questions are addressed in Chapter 4.

Both FcγRIIA and FcγRIIB2 are expressed by human myeloid cells and possess endocytic capacity. They have homologous extracellular domains and can be simultaneously co-engaged by multivalent immune complexes. The question of how co-engagement of both receptors affects their internalization and the following intracellular sorting is addressed in both

Chapter 3 and Chapter 4.

In this thesis, I investigate the trafficking behaviours of human FcγRIIA and FcγRIIB2.

Studies were carried out in 1) a transfected cell model heterologously expressing either or both receptors, and 2) primary human myeloid cells including freshly isolated monocytes and

cytokine differentiated macrophages or iDCs. Chapter 3 of this thesis describes the intracellular sorting events of FcγRIIA and FcγRIIB2 following their internalization via endocytosis. The final fates of receptors and immune complexes as well as the endocytic compartments they are sorted into are examined when they are engaged in isolation or upon co- engagement. This chapter also investigates the molecular mechanism important for the intracellular transport of FcγRIIA and FcγRIIB2. In Chapter 4, I compare the requirements for ubiquitylation and the internalization kinetics of these two endocytic pathways. I also probe how co-engagement of FcγRIIA and FcγRIIB2 affects the kinetics of their internalization.

This thesis provides insights into how antigens in immune complexes internalized via potentially multiple FcγR subtypes are handled by human myeloid cells and the effects of different properties of FcγRs on deciding alternative fates of receptors and immune complexes.

Receptor trafficking and antigen processing are closely associated with receptor signalling and antigen presentation. Therefore, divergence in the trafficking of FcγRIIA and FcγRIIB2 during endocytic trafficking can have impacts on a number of FcγR-mediated responses. These include modulation of the duration and magnitude of activating receptor signalling and inflammatory responses, the overall balance of activating and inhibitory receptors on the cell surface and antigen presentation. This study will help us understand more about immune complex-mediated host defence against foreign pathogens. It also provides theoretical background which opens up novel and potentially promising therapeutic approaches to control the exacerbation of autoimmune diseases.

Chapter 2

Materials and Methods

2.1 Reagents and antibodies

Fetal bovine serum, α-minimal essential medium, Roswell Park Memorial Institute medium (RPMI) 1640 and G418 were from Wisent (St. Bruno, Quebec, Canada). Mouse anti- ubiquitin antibody P4G7 and mouse anti-myc antibody 9E10 were from Covance (Berkeley,

CA, USA). Rabbit anti-myc antibody A14, mouse anti-phosphotyrosine antibody 4G10, goat anti-EEA1 antibody, goat antibodies against FcγRIIA and FcγRIIB intracellular domain (goat anti-IIA, goat anti-IIB) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Mouse anti-FcγRIIA extracellular domain antibody IV.3 was purified from hybridoma supernatants and Fab fragments were prepared using a Pierce ImmunoPure Fab Kit (Thermo Fisher

Scientific, Rockford, IL, USA). Alexa488-IV.3 was prepared using Alexa488 protein labelling kit (Invitrogen, Eugene, OR, USA). Mouse anti-FcγRII extracellular domain antibody AT10 was from Abcam (Cambridge, MA, USA). Mouse anti-FcγRIIB extracellular domain antibody

4F5 was a kind gift of Dr. Robert Kimberly (University of Alabama, Birmingham, AL, USA).

Rabbit anti-hamster LAMP1 antibody UH1 was from the Developmental Studies Hybridoma

Bank (University of Iowa, Iowa City, IA, USA). Cy3-, Cy5-, and horseradish peroxidase- conjugated secondary antibodies were from Jackson Immunoresearch Laboratories (West

Grove, PA, USA). Alexa488-conjugated secondary antibodies, Alexa647-dextran, Alexa488- transferrin and Rhodamine-transferrin were from Invitrogen (Eugene, OR, USA).

Paraformaldehyde was from Canemco (16%, EM grade). PP1 was from Biomol International

(Plymouth Meeting, PA, USA). LY294002 and MG132 were from CalBiochem (San Diego,

CA, USA). GM-CSF and cycloheximide were from Bioshop (Burlington, Ontario, Canada). 35

IL-4 and IL-10 were from BioSource International (Camarillo, CA, USA). Supersignal West

Pico chemiluminescent substrate, restore western blot stripping buffer, and ultralink protein G beads were from Thermo Scientific (Rockford, IL, USA). Sheep red blood cells and rabbit anti-sheep red blood cell antibody were from ICN biomedical (Irvine, CA, USA). Human IgG

(catalog #I4506), protease inhibitors, anti-β actin antibody, and other chemicals were from

Sigma-Aldrich (St. Louis, MO, USA). Mouse anti-CD64-FITC and IgG1k-FITC were from BD

Pharmingen (BD Biosciences, San Diego, CA, USA).

2.2 Cell culture

ts20 cells, a Chinese hamster fibroblast cell line with a temperature sentitive mutation in its E1 ubiquitin activating enzyme, were grown at 34°C (necessary for the growth of the cells due to the temperature sensitive defect) and 5% CO2 in α-minimal essential medium + 10% fetal bovine serum. THP-1 cells were cultured at 37oC in RPMI 1640 containing 10mM

HEPES, 1mM sodium pyruvate, 0.05 mM 2-mercaptoethanol, 2mM L-glutamine, 4.5g/L glucose, 1.5g/L sodium bicarbonate with 10% FBS,

For preparation of primary human monocyte-derived macrophage or moDC, peripheral blood mononuclear cells were isolated from blood of healthy donors by Ficoll-Paque Plus (GE

Healthcare, Pittsburgh, PA, USA) density centrifugation, followed by isolation of monocytes by adherence to tissue culture plastic. In some experiments, human monocytes were isolated from peripheral blood of healthy donors by monocyte enrichment by negative selection using

RosetteSep Monocyte Enrichment Cocktail (StemCell Technologies, Vancouver, British

Columbia, Canada) according to the manufacturer’s instructions. Monocytes were cultured overnight at 37oC at 0.5 ¯106 cells/ml in RPMI with 30ng/ml of both IL-4 and IL-10 for upregulation of FcγRIIB expression before analysis (Wijngaarden et al., 2004). To generate

monocyte-differentiated macrophages, freshly isolated peripheral blood mononuclear cells were loaded to the center of glass coverslips and incubated in warm medium for 1.5 hour to allow for the adherence of monocytes to glass coverslips. The non-adherent lymphocytes were then removed and the remaining monocytes were cultured for 6 days in RPMI + 10% fetal bovine serum with GM-CSF (1000 U/ml) to induce differererntiation to macrophages. Isolated monocytes were cultured with GM-CSF (1000 U/ml) and IL-4 (1000 U/ml) for 6 days to allow for differentiation into moDC after removal of non-adherent lymphocytes. Monocyte-derived macrophages were identified by their size and spread morphology; moDCs were identified based on their size and dendritic morphology.

2.3 DNA constructs and transfection

cDNAs for wild-type or mutated forms of FcγRIIA (His131 variant) and FcγRIIB2 were cloned into pcDNA3.1/MycHis or pcDNA3.1/His (Invitrogen, Carlsbad, CA, USA) for expression with C-terminal MycHis (MH) or His tags. Chimeric FcγRIIA/FcγRIIB2 MH- tagged receptors were generated by PCR amplification of receptor sequences from GFP-tagged chimeric receptors (Syam et al., 2010) and cloning into pcDNA3.1/MycHis. FcγRIIA mutant

“IIAΔC”, “IIA M288AΔC” and “IIA G286AΔC” were generated by PCR from wildtype MH tagged FcγRIIA (FcγRIIA-MH). Site-directed mutations “IIA E281AΔC”, “IIA T282AΔC”,

“IIA D284AΔC”, “IIA G285AΔC”, “IIA G286AΔC” and “IIA N291AΔC” were generated using the QuikChangeTM Mutagenesis Kit (Stratagene, La Jolla, CA) with “IIAΔC” as the template. The following oligonucleotides were used as primers for mutagenesis. Lower case letters denote mutated bases. The underlined bases denote restriction sites incorporated immediately downstream of the last amino acid of FcγRIIA when generating the truncation mutant.

FcγRIIA mutants generated by PCR:

IIAΔC: Forward primer: 5’ G GGA GAA ACC ATC ATG CTG AG 3’

Reverse primer: 5’ GGG TCT AG aTT CAG AGT CAT GTA GC 3’

IIA M288AΔC: Forward primer: 5’ G GGA GAA ACC ATC ATG CTG AG 3’

Reverse primer: 5’ GGG TCT AG aTT CAG AGT ggc GTA GC 3’

IIA L290AΔC: Forward primer: 5’ G GGA GAA ACC ATC ATG CTG AG 3’

Reverse primer: 5’ GGG TCT AG aTT Cgc AGT CAT GTA GC 3’

FcγRIIA mutants generated by site directed mutagenesis:

IIA E281AΔC: 5’ CC AAC AAT GAC TAT GcA ACA GCT GAC GGC GGC TAC 3’

IIA T282AΔC: 5’ C AAC AAT GAC TAT GAA gCA GCT GAC GGC GGC TAC 3’

IIA D284AΔC: 5’ GAC TAT GAA ACA GCT GcC GGC GGC TAC ATG ACT CTG 3’

IIA G285AΔC: 5’ GAA ACA GCT GAC GcC GGC TAC ATG ACT CTG 3’

IIA G286AΔC: 5’ GAA ACA GCT GAC GGC GcC TAC ATC ACT CTG 3’

IIA N291AΔC: 5’ C GGC TAC ATG ACT CTG gcT CTA GAG GGC CCG CGG 3’

The complementary oligonucleotides were used in combination with these primers for the above mutants generated by site directed mutagenesis.

All mutations were confirmed by DNA sequencing. Rab11-GFP was a kind gift from Dr.

Rene Harrison (University of Toronto at Scarborough, Scarborough, Ontario, Canada). MH tagged wildtype FcγRIIA and FcγRIIB2 as well as FcγRIIA mutants “IIA Y287F” and “IIA

T289A” were kind gifts from Dr. Alan D. Schreiber (University of Pennsylvania, Philadelphia,

PA, USA). 2-FYVE-GFP (two tandem FYVE domains attached to GFP) were provided by Dr.

Sergio Grinstein ((University of Toronto, Toronto, Ontario, Canada).

Transfections into ts20 cells were performed with FuGENE 6 (Roche Applied Science) or

Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturers'

instructions. Stable cell lines expressing FcγRIIA, FcγRIIB2 and mutant receptors were selected with G418 (0.5mg/ml).

2.4 Ubiquitylation abolishment and inhibitor treatment

To abolish cellular ubiquitylation, the E1 ubiquitin activating enzyme was inactivated by pre-incubating ts20 cells at a non-permissive temperature (42.5oC) for 2 hours. For treatment of cells with inhibitors, cells were pre-treated for 30 minutes (PP1, LY294002, bafilomycin A1),

1 hour (cycloheximide) or 2 hours (MG132) by adding inhibitor directly to the culture medium at 30 μM (PP1), 20 μM (LY294002), 300 nM (bafilomycin A1), 100 μg/ml (cycloheximide) or

20 μM (MG132).

2.5 Development of rabbit polyclonal antibodies specific to the intracellular domains of FcγRIIA or FcγRIIB2

The intracellular domains of human FcγRIIA (residues T250-N316) or FcγRIIB2

(residues N257-I290) were individually cloned into the pGEX-3X vector by PCR to express the domains as fusion proteins to glutathione S-transferase (GST). The following oligonucleotides were used for DNA cloning. The underlined bases denote restriction sites incorporated when

PCR was performed:

FcγRIIA: Forward primer: 5’ T GGATCC CC ACT GAT CCT GTG AAG GCT T 3’

Reverse primer: 5’ CCT GAATTC TTA GTT ATT ACT GTT GAC 3’

FcγRIIB2: Forward primer: 5’ T GGATCC CC AAT CCT GAT GAG GCT GAC 3’

Reverse primer: 5’ CCT GAATTC CTA AAT ACG GTT CTG GTC 3’

The GST-tagged proteins were expressed in BL21 bacteria, purified on glutathione- agarose and used for immunizations of New Zealand white rabbits (Division of Comparative

Medicine, University of Toronto, Toronto, Ontario, Canada). Antisera were taken from the rabbit after three rounds of immunization and anti-GST antibodies contained in the antisera were removed from antisera using GST-agarose.

2.6 Endocytosis and phagocytosis assays

Human IgG (10 mg/ml) was aggregated at 62oC for 20 minutes followed by centrifugation at 16,000×g for 10 minutes to precipitate insoluble IgG aggregates. Supernatants containing soluble aggregates were used at 1:100 dilution to induce endocytosis by incubating receptor-expressing ts20 cells at 34oC for indicated times. In experiments where FITC conjugated-heat aggregated IgG (FITC-agIgG) was used to trigger endocytosis, FITC was conjugated to IgG (approximately 4 FITC per IgG), then FITC-IgG was mixed with unlabeled

IgG at a ratio of 1:4 before heat aggregation. Cells were incubated with FITC-agIgG for 20 minutes on ice before transfer to warm medium for initiate endocytosis. In experiments where endocytosis was triggered with anti-receptor antibodies, IV.3 (anti-FcγRIIA) or AT10 (pan anti-

FcγRII) were added to transfected ts20 cells, THP-1 or primary human myeloid cells at 0.5

μg/ml for 20 minutes at 4oC. After washing, cells were incubated for 20 minutes at 4oC with 1

μg/ml Cy5- donkey anti-mouse antibody or Alexa488 goat anti-mouse antibody to cross-link the receptors, followed by warming to 34oC (ts20 cells) or 37oC (THP-1 or primary monocytes) to trigger endocytosis. To measure the surface expression of FcγRIIA and FcγRIIB in different human myeloid cells, cells were immediately subjected to fixation and analyzed by microscopy after incubation with IV.3 or 4F5 (anti-FcγRIIB) antibodies followed by secondary cross- linking as above. To assess FcγRIIA-mediated internalization in moDCs by flow cytometry, cells were incubated with Alexa488-IV.3 on ice for 20 minutes before warming the medium up to 37oC to trigger endocytosis. In some experiments where subsequent immunofluoresence was

performed using mouse antibodies, isotype-specific secondary antibodies were employed. In moDC, for transferrin loading, rhodamine- or Alexa488-conjugated transferrin was added at

50μg/ml for the last 10 minutes of incubation. For dextran loading, Alexa647-conjugated dextran was added at 50 μg/ml for 1 hour followed by a chase for 1 hour (ts20 cells, macrophages, moDCs) or overnight (monocytes) at 37oC.

For phagocytosis assays, sheep red blood cells were opsonized with rabbit anti-sheep red blood cell antibody (1:50 dilution) in PBS for 1 hour at room temperature. ts20 cells expressing

FcγRIIA grown on glass coverslips were incubated with opsonised sheep red blood cells at

34 °C for 40 minutes and washed. The sheep red blood cells that were not internalized were then lysed by a brief exposure to water before the samples were further processed for immunofluorescence staining.

2.7 Microscopy and immunofluorescence

Cells were washed and fixed with 4% paraformaldehyde for 30 minutes, then permeabilized with 0.1% Triton X-100 at room temperature for 20 minutes. In some experiments, agIgG remaining on the cell surface was detected by incubating cells on ice for 10 min with fluorescently labelled anti-human secondary antibodies before fixation. To detect

FcγR, cells were first blocked with 5% BSA in phosphate buffered saline (PBS) or 5% donkey serum for goat antibodies. They were then incubated with anti-myc antibody 9E10, anti-

FcγRIIA antibody or anti-FcγRIIB2 antibody at 1:1000 dilution in blocking buffer for one hour.

For EEA1 immunofluoresence, cells were incubated with anti-EEA1 antibody at 1:50 dilution in 5% donkey serum for one hour after the blocking step. For LAMP1 immunofluorescence, cells were permeabilized with methanol at -20oC for 20 minutes before they were blocked with

5% BSA and treated with UH1 antibody at 1:1 dilution. Samples were then treated with fluorophore-conjugated secondary antibodies at 0.8μg/ml in PBS for 30 minutes before they 41

were mounted with DAKO mounting medium for microscopy analysis. Total agIgG in the cells was detected with fluorophore-conjugated anti-human secondary antibody. For sheep red blood cell phagocytosis assay, sheep red blood cells remaining outside the ts20 cells were labeled with Cy3 anti-rabbit antibody. After fixation with 4% paraformaldehyde and permeablization with 0.1% Triton X-100, total sheep red blood cells both outside and inside the ts20 cells were detected by Cy5 anti-rabbit antibody.

Fixed and mounted cells were analyzed by widefield microscopy using a Zeiss Axiovert

200M microscope with 40× or 100× oil immersion objectives or by confocal microscopy using a Zeiss LSM 510 confocal scanning microscope with a 63× objective. Alexa488, Cy3 and Cy5 signals were detected using standard filter sets. For each sample, observations were made based on analysis of seven to eight fields of cells of approximately same number of cells per field to ensure the consistency of results among cells. Only when at least 70 percent of the cells of interest in each field showed similar pattern of behaviour was a conclusion reached.

2.8 Flow cytometry

ts20 cells expressing MH-tagged FcγRIIA or FcRIIB2 were detached from culture dishes and dispersed in PBS. Following fixation with 2% paraformaldehyde and permeabilization with 0.1% Triton X-100, cells were blocked with 5% BSA and stained with anti-myc antibody

9E10 at 1:1000 dilution in blocking buffer for 30 minutes at room temperature. After washing, cells were stained with Alexa488-conjugated anti-mouse antibody (0.8μg/ml for 15 minutes) and analyzed by flow cytometry using a FACSCalibur (Becton Dickinson, Franklin Lakes, NJ,

USA). For the detection of aggregated IgG, cells were stained with 1.5 μg/ml Cy5-anti-human secondary antibody. In some experiments, receptor-expressing ts20 cells were stained with

Cy5 donkey anti-goat antibodies or Cy5 anti-mouse antibody (2 μg/ml for 15 minutes) after

fixation to detect the remaining antibodies on the cell surface. Monocytes were incubated with

Cy5 anti-mouse antibodies on ice for 10 minutes before fixation. Cy5 fluorescence was analyzed by flow cytometry using a FACSCalibur (Becton Dickinson). For measurement of internalization of FITC-agIgG by ts20 transfectants, surface FITC-agIgG was quenched with

0.1% trypan blue solution for 10 minutes at room temperature before fixation of the cells and analysis with flow cytometry.

2.9 Immunoprecipitation and Western Blotting

To detect FcγRII or antibody complexes, ts20 or THP-1 cells were lysed in lysis buffer

(1% Triton X-100, 0.1% SDS, 0.5% deoxycholate, 1 mM NaF, 0.1% protease inhibitor mixture in PBS). 2mM Na3VO4 and 10μM phenylarsine oxide were added into the lysis buffer when blotting for receptor phosphorylation. Lysis buffer used for receptor ubiquitylation detecting experiments also contained 20 mM NEM. Lysates were then incubated on ice for 20 minutes.

Insoluble material was removed by centrifugation at 16,000×g for 10 minutes at 4oC and lysates were frozen for future analysis.

When immunoprecipitation was required before analysis of samples by western blotting,

1 μg of IV.3 or A14 antibody and 25 μl of protein G beads were mixed with cell lysates followed by overnight nutation at 4oC. After washing beads two times with lysis buffer and two times with PBS-T (PBS with 0.05% Tween 20), beads were resuspended in Laemmli’s sample buffer. For receptor ubiquitylation or phosphorylation detection experiments, the beads were washed three times with TBS-T (TBS containing 0.1% Tween 20) instead before resuspension in Laemmli’s sample buffer.

Samples were resolved by SDS-PAGE, transferred to nitrocellulose membrane (Bio-Rad,

Hercules, CA, USA), blocked with 5% BSA in PBS-T for 1 hour at room temperature and

probed with primary antibody (anti-myc antibody 9E10, anti-FcγRIIA or anti-FcγRIIB antibodies) at 1:1000 dilution in blocking buffer overnight. Blots were washed in PBS-T three times, incubated with anti-mouse or anti-rabbit horseradish peroxidase for 30 minutes, washed again and developed with Supersignal West Pico chemiluminescent substrate (Thermo

Scientific, Rockford, IL, USA). A Genius2 Bioimager (Syngene, Frederick, MD, USA) was used to visualize the blots. Blots were stripped between probings using restore western blot stripping buffer. For blotting for phosphotyrosine, TBS-T instead of PBS-T was used for blocking, washing and primary antibody (anti-ubiquitin antibody)/secondary antibody probing steps. CL-Xposure film (Thermo Scientific, Rockford, IL, USA) was used to visualize the blots.

Chapter 3 Divergent Intracellular Sorting of FcγRIIA and

FcγRIIB2

* Fig. 3-1,Fig. 3-2, Fig. 3-6, Fig. 3-8 to Fig. 3-11, Fig. 3-14, Fig. 3-17, Fig. 3-20 to Fig. 3-22 in this chapter was published in Zhang CY and Booth JW. Divergent Intracellular Sorting of

FcγRIIA and FcγRIIB2. J Biol Chem. 285(44):34250-8

* Fig 3-3 to Fig. 3-5, Fig. 3-7, Fig. 3-12, Fig. 3-13, Fig. 3-15, Fig. 3-16, Fig. 3-18, Fig. 3-19,

Fig. 3-23 are unpublished data

3.1 Abstract

The human low affinity FcγRII family includes both the activating receptor FcγRIIA and the inhibitory receptor FcγRIIB2. These receptors have opposing signalling functions but are both capable of internalizing IgG-containing immune complexes through clathrin-mediated endocytosis. I demonstrate that upon engagement by multivalent aggregated human IgG,

FcγRIIA expressed in ts20 Chinese hamster fibroblasts is delivered along with its ligand to lysosomal compartments for degradation, while FcγRIIB2 dissociates from the ligand and is routed separately into the recycling pathway. FcγRIIA sorting to lysosomes requires receptor multimerization, but does not require either SFK activity or ubiquitylation of receptor lysine residues. The sorting of FcγRIIB2 away from a degradative fate is not due to its lower affinity for IgG and occurs even upon persistent receptor aggregation. Upon co-engagement of

FcγRIIA and FcγRIIB2, the receptors are sorted independently to distinct final fates after

dissociation of co-clustering ligand. These results reveal fundamental differences in the trafficking behaviours of different Fcγ receptors.

3.2 Introduction

FcγRs are key players in immune responses. Widely expressed on cells of the hematopoietic system, these receptors mediate a multitude of biological responses that are triggered upon receptor engagement by multivalent IgG-containing immune complexes

(Nimmerjahn et al., 2008). These responses include production of inflammatory cytokines, antibody-dependent cellular cytotoxicity and induction of dendritic cell maturation. FcγRs also mediate the internalization of immune complexes. Soluble immune complexes are internalized by clathrin-mediated endocytosis while large (>0.5 µm) antibody-coated particles are internalized via phagocytosis. These uptake processes are important in host defence against infection. Moreover, defects in immune complex clearance are associated with the development of autoimmunity (Takai, 2002).

FcγRs can be categorized into two functional groups. The activating FcγRs have ITAMs within the cytoplasmic domain of the receptor itself or an associated Fc receptor-γ chain signalling subunit. Phosphorylation of tyrosine residues in the ITAMs by SFK after receptor aggregation initiates signalling cascades that trigger downstream effector responses (Daeron,

1997). In contrast, inhibitory FcγRs contain ITIMs, which recruit phosphatases that antagonize

ITAM-mediated signalling. Therefore, the responsiveness of effector cells to immune complexes is determined by the balance between activating and inhibitory receptors

(Nimmerjahn et al., 2008).

A feature of the human immune system that distinguishes it from that of mouse is the presence of both activating and inhibitory members of the low affinity FcγRII subfamily.

FcγRIIA is an activating FcγRs unique to humans and other primates and is the most widely expressed FcγR on human leukocytes (Tan Sardjono et al., 2003). In addition, FcγRIIA is unusual among activating FcγR in having both ligand binding and ITAM signalling domains contained in a single polypeptide chain. FcγRIIA exists as two codominantly expressed polymorphic variants, with either histidine or arginine at residue 131. The His131 form has a higher affinity for several IgG subclasses (Bruhns et al., 2009). FcγRIIA can mediate phagocytosis of large IgG-opsonized particles, which depends on ITAM-mediated signalling and rearrangements of the actin cytoskeleton (Indik et al., 1991). FcγRIIA is also able to mediate clathrin-dependent endocytosis of soluble IgG-containing immune complexes (Budde et al., 1994; Mero et al., 2006).

Human leukocytes also express the inhibitory receptor FcγRIIB. While the extracellular domains of FcγRIIA and FcγRIIB have 92% amino acid identity, their intracellular domains are divergent, with FcγRIIB containing an ITIM. Two different isoforms of FcγRIIB can be generated through alternative splicing. FcγRIIB1 is expressed mainly in B cells, where it inhibits signalling from the BCR when the two receptors are coengaged by antigen-antibody complexes (Ravetch et al., 2001). FcγRIIB2 is expressed mainly in myeloid cells. Human

FcγRIIB2 lacks the ability to support phagocytosis of IgG-opsonised large particles. On the contrary, it negatively regulates signalling for phagocytosis and other ITAM-dependent responses when coengaged with activating FcγR (Hunter et al., 1998). FcγRIIB2 is, however, able to mediate endocytosis of soluble immune complexes due to the presence of a di-leucine motif in its cytoplasmic domain (Hunziker et al., 1994; Matter et al., 1994; Van den Herik-

Oudijk IE et al., 1994).

As both of these receptors can mediate immune complex uptake, I sought to investigate how they traffic within the cell after such internalization. This is important for understanding

both the overall regulation of inflammatory signalling as well as the processing of immune complexes internalized via FcγRs. I demonstrate that, in addition to their opposing effects on cell activation, FcγRIIA and FcγRIIB2 exhibit distincttrafficking behaviours after internalization, with FcγRIIA but not FcγRIIB2 being targeted for degradation.

3.3 Results

3.3.1 Aggregated IgG internalized by either FcγRIIA or FcγRIIB2 is degraded in lysosomes

To better understand how these two FcγRs traffic, FcγRIIA or FcγRIIB2 carrying MH tags were stably transfected into ts20 cells, a Chinese hamster fibroblast cell line. This heterologous expression system allows for the study of the trafficking behaviour of a single class of FcγR either in its wild type or mutated forms (Booth et al., 2002; Mero et al., 2006;

Syam et al., 2010). Soluble complexes of heat aggregated IgG (agIgG), a mimic of soluble multivalent immune complexes, were used to trigger endocytosis of either FcγRIIA or

FcγRIIB2. Both receptors mediated uptake of agIgG (Fig. 3-1B, E). The agIgG extensively co-localized with LAMP1 by 80 minutes, suggesting the delivery of agIgG to late endosomal/lysosomal compartments (Fig. 3-1A-F). No significant uptake of agIgG was observed in untransfected cells (data not shown). Consistent with the observed delivery to lysosomes, agIgG internalized via either receptor underwent degradation (Fig. 3-1G).

3.3.2 FcγRIIA, but not FcγRIIB2, undergoes degradation in lysosomes after internalization

While agIgG ligand internalized by either FcγRIIA or FcγRIIB2 underwent similar degradation, a second important question is the fate of the receptors themselves. To address

LAMP1 agIgG LAMP1/agIgG

ABC

D E F D E F

G agIgG 120 IIA

100 I

F IIB2

l M l

ia t

i 60

in f

o 40 % 20 0 0100200 time (min)

Fig. 3-1 agIgG internalized by either FcγRIIA or FcγRIIB2 is degraded in lysosomes. (A- F) ts20 cells expressing MH-tagged FcγRIIA (A-C) or FcγRIIB2 (D-F) were incubated with agIgG for 80 minutes. agIgG was detected by immunofluorescence with anti-human IgG antibody (B, E). LAMP1 was localized with anti-LAMP1 antibody (A, D). Merged images show agIgG in green and LAMP1 staining in magenta (C, F). White in the merged images reflects co-localizations of LAMP1 and agIgG; co-localization analysis by fluorescence microscopy is discussed in further detail in the Appendix at the end of this chapter (pg. 96-99).. Arrows highlight instances of co-localization of agIgG and LAMP1. Scale bar: 10 μm. Images were analyzed by widefield microscopy. Representative of three experiments. (G) ts20 cells expressing FcγRIIA or FcγRIIB2 were incubated with agIgG at 4oC, washed, and chased at 34oC for the indicated times. Cells were fixed and permeabilized. Total agIgG remaining was detected with Cy5- anti-human IgG antibody and quantified by flow cytometry. agIgG level is expressed as a percentage of the initial mean fluorescence intensity (MFI) at time 0 after background subtraction of fluorescence intensity of untransfected ts20 cells incubated with agIgG and stained with Cy5- anti-human IgG antibody. Triangles: FcγRIIA; squares: FcγRIIB2. Error bars indicate s.d. n = 3. 49

this, immunoblotting was performed to determine the amount of receptors remaining in cells after stimulation with agIgG. FcγRIIA and FcγRIIB2 appear as doublets in western blotting, possibly due to different glycosylation of the receptors (Fig. 3-2A). For FcγRIIA, receptor levels declined after induction of endocytosis, such that by 5 hours after addition of agIgG very little FcγRIIA remained (Fig. 3-2A). In contrast, little if any drop in FcγRIIB2 levels was observed over the same time frame. The total amounts of FcγRIIA and FcγRIIB2 were also quantified by flow cytometry using intracellular staining with anti-myc antibody. The relative expressions of the two receptors are similar in the ts20 cells that are stably transfected with either one of the receptors (Fig 3-2B). Fig 3-2C showed similar loss of FcγRIIA but not

FcγRIIB2. FcγRIIB2 does not undergo rapid protein turnover as FcγRIIB2 level does not change after treatment with cycloheximide for up to 5 hours (Fig. 3-3A). No drop in the

FcγRIIB2 level was observed after agIgG stimulation even in the presence of cycloheximide, indicating that the persistence of FcγRIIB2 following endocytosis is not due to new protein synthesis (Fig. 3-3B, C). Similar differential sorting of FcγRIIA and FcγRIIB2 was also observed in ts20 cells expressing untagged receptors, arguing the difference is not due to effects of the MH tag (Fig. 3-4).

The loss of FcγRIIA after endocytosis was also analyzed in THP-1 cells, a human monocytic cell line that expresses endogenous FcγRIIA. To detect endogenous FcγRIIA in human myeloid cells, a rabbit antibody against the intracellular domain of FcγRIIA was developed and its specificity was tested in receptor-expressing ts20 cells, THP-1 cells and

Ramos cells by western blotting and immunofluorescence (Fig. 3-5). As in the ts20 transfectants, FcγRIIA largely disappeared in the THP-1 cells within 3 hours of treatment with agIgG (Fig. 3-2D). This loss was inhibited by treatment with bafilomycin, an inhibitor that blocks endosomal acidification, indicating that degradation occurs in the endo/lysosomal

A receptor FcγRIIA FcγRIIB2 4 C 3hr 5hr 4 C 3hr 5hr agIgG - +++- + ++ 55 IIA IIB2 43 anti-myc

β-actin B C receptor level 120 100 100

I I

x x

F F a a 80

m m

l M M l l f

f 40 60

ia ia

o o

it 40 %

% 20

in in f

0 f 20

o o

100 101 10234 10 10 % % 0 fluorescence 0100200 time (min) D IP IV.3+ - bafilomycin - - - + - agIgG (min) - 30 180 180 -

34 26

Fig. 3-2 FcγRIIA, but not FcγRIIB2, undergoes degradation after internalization. (A) Degradation of MH-tagged receptors expressed in ts20 cells was followed after addition of agIgG for 3 or 5 hours at 34oC, or for 20 minutes at 4oC. Receptor levels were analyzed by western blotting with anti-myc antibodies. The blot was stripped and reprobed with β-actin antibody. Representative of five experiments. (B) Anti-myc staining of permeabilized untransfected ts20 cells (shaded histogram) or ts20 cells expressing FcγRIIA-MH (solid line) or FcγRIIB2-MH (dashed line) was analyzed by flow cytometry. (C) Loss of MH-tagged receptors expressed in ts20 cells after addition of agIgG was measured by anti-myc staining and flow cytometry. Receptor level is expressed as a percentage of the initial mean fluorescence intensity after subtraction of background anti-myc staining of untransfected ts20 cells. Open triangles: FcγRIIA; closed squares: FcγRIIB2. Error bars indicate s.d. n = 3. (D) agIgG was added to 107 THP-1 cells for indicated times at 37oC in the presence or absence of bafilomycin. FcγRIIA was immunoprecipitated from cell lysates with IV.3 before western blotting with rabbit anti-FcγRIIA antibody. Thick arrow indicates FcγRIIA. Arrowhead indicates immunoprecipitating antibody light chain. Thin arrow indicates non-specific protein in the lysates that binds to protein G beads. Representative of five experiments.

A receptor none FcγRIIB2 CXH - - 1hr 2hr 3hr 4hr 5hr

anti-myc (receptor)

β-actin B receptor none FcγRIIB2 CXH -+ 1hr 2hr 3hr 1hr 2hr 3hr agIgG -- +++- +++ 43

anti-myc (receptor)

β-actin C

total receptor level (anti-myc) 160

140 IIB2 -CXH

I I F

F 120 IIB2 +CXH l M l

l M l 100

ia ia it

it 80

in in f

f 60 o

o 40

% % 20 0 01234 agIgG uptake (hr)

Fig. 3-3 The effect of cycloheximide on the level of FcγRIIB2 with or without ligand stimulation. FcγRIIB2-MH expressing ts20 cells were treated with or without cyclohexamide (CXH) for different lengths of time without any ligand stimulation (A). Alternatively, these cells were stimulated with agIgG for different lengths of time after pre-treatment with or without CXH for 1 hour (B, C). (A, B) The total cell lysates were blotted with anti-myc antibody by western blotting. The blot were stripped and reprobed with anti-β-actin antibody as a loading control. Representative of two experiments. (C) The level of remaining receptor was measured by intracellular staining with anti-myc antibody and flow cytometry to follow the decrease in mean fluorescence intensity. Squares: without cycloheximide; diamonds: with cycloheximide. The total receptor level measured at each time point is expressed as the percentage of the initial mean fluorescence intensity of stained agIgG at 0 minute. Preliminary experiment. 53

A receptor none FcγRIIA-MH FcγRIIA agIgG + - +++- +++ time (min) 40- 40 90 180 - 40 90 180 55

IP: IV.3 WB: anti-FcγRIIA 55

IP: IV.3 WB: anti-myc

B receptor none FcγRIIB2-MH FcγRIIB2 agIgG + - +++ - +++ time (min) 40- 40 90 180 - 40 90 180

WB: anti-FcγRIIB

WB: anti-myc

WB: β-actin

Fig. 3-4 MH tag does not affect the intracellular sorting of FcγRIIA- or FcγRIIB2- mediated endocytosis. agIgG was added to ts20 cells expressing MH-tagged or untagged FcγRIIA (A), ts20 cells expressing MH-tagged or untagged FcγRIIB2 (B) or untransfected cells followed by incubation at 34oC for indicated lengths of time. Lysates were analyzed by western blotting with goat anti-IIA following immunoprecipitation with IV.3 antibody (A) or western blotting with goat anti-FcγRIIB antibody (B). The blot was stripped and reprobed with anti-myc antibody and then with β-actin antibody. Thick arrow indicates MH tagged- or untagged-FcγRIIA. Double headed thin arrow indicates immunoprecipitating antibody heavy chain. Representative of three experiments.

A anti-FcγRIIA sera anti-FcγRIIB2 sera receptor: none IIA-MH IIB2-MH none IIA-MH IIB2-MH 55 IIA IIB2

B anti-FcγRIIA anti-FcγRIIA+IIA ptn anti-FcγRIIA (GST cleaned) Cells: THP1 Ramos THP1 Ramos THP1 Ramos

36 IIA cross-reacting

anti-FcγRIIB anti-FcγRIIB+IIB ptn anti-FcγRIIB (GST cleaned) Cells: THP1 Ramos THP1 Ramos THP1 Ramos

36 IIB2 cross-reacting

receptor expressed IIA-MH IIB2-MH RIIB RIIA γ γ anti-Fc anti-Fc CE anti-sera anti-myc anti-myc

D F

Fig. 3-5 Rabbit anti-FcγRIIA sera and anti-FcγRIIB sera. (A,B) FcγRIIA-MH or FcγRIIB2-MH expressing ts20 cells (A), THP-1 (IIA+IIB-) or Ramos (IIA-IIB+) (B) cells were lysed and analyzed by western blotting with rabbit anti-FcγRIIA or anti-FcγRIIB sera. In some samples, to confirm the specificity of the anti-sera, anti-FcγRIIA sera or anti-FcγRIIB sera were pre-mixed with FcγRIIA or FcγRIIB2 proteins (IIA ptn, IIB ptn) respectively before being used for immunoblotting. To minimize the background, anti-GST antibodies in the anti-sera were removed by mixing the anti-sera with GST-bound agarose beads (GST cleaned) before being used for western blotting analysis. Representative of two experiments. (C-F) FcγRIIA-MH (C, D) or FcγRIIB2-MH (E, F) in ts20 cells were localized by immunofluorescence with anti- FcγRIIA sera (C), anti-FcγRIIB sera (E) or anti-myc 9E10 antibodies (D,F). Images were analyzed by widefield microscopy. Representative of two experiments. 55

system.

To characterize the subcellular localization of FcγRIIA and FcγRIIB2 after endocytosis, the tagged receptors were detected by immunofluorescence. Under unstimulated conditions, both FcγRIIA and FcγRIIB2 were observed to reside on the cell surface and in diffuse perinuclear vesicles (Fig. 3-6A, J). This intracellular compartment is a recycling endosome pool, as judged by its co-localization with loaded transferrin (Fig. 3-6A-C, J-L). After 80 minutes of agIgG treatment, FcγRIIA was largely co-localized with internalized agIgG ligand, which, as noted above (Fig. 3-1), has moved by this time into punctate perinuclear lysosomes

(Fig. 3-6G-I). These puncta, while located in the central region of the cell like the transferrin- positive recycling endosomes, are adjacent to these endosomes rather than co-localized with them (Fig. 3-6D-F). Thus, consistent with the observed receptor degradation, FcγRIIA is targeted to lysosomes along with its ligand. In contrast, at the same time point after agIgG addition, FcγRIIB2 showed a localization similar to that seen in unstimulated cells, namely, it was found at the cell surface and in transferrin-positive endosomes (Fig. 3-6M-O), and did not follow internalized agIgG to lysosomes (Fig. 3-6P-R), consistent with the observed lack of

FcγRIIB2 degradation. To confirm that FcγRIIB2 is sorted into recycling endosomes after internalization, GFP-tagged Rab11, a protein marker of recycling endosomes, was transiently transfected into ts20 cells stably expressing FcγRIIA or FcγRIIB2. In agreement with findings from the transferrin co-localization experiment, FcγRIIA is sorted into Rab11-negative lysosomal compartments after 80 minutes of agIgG engagement whereas a large fraction of

FcγRIIB2 is delivered into Rab11-containing diffuse perinuclear recycling endosomes (Fig. 3-

7E-H, M-P). Thus, FcγRIIB2 is sorted to recycling endosomes while agIgG ligand internalized via this receptor is degraded in lysosomes.

FcγRIIA receptor Tf Tf/receptor

0’

ABC

80’ DEF receptor agIgG agIgG/receptor

80’

GH I FcγRIIB2 receptor Tf Tf/receptor

0’

JKL

80’

M N O receptor agIgG agIgG/receptor

80’

P Q R

Fig. 3-6 FcγRIIA, but not FcγRIIB2, follows agIgG to lysosomes. ts20 cells expressing MH- tagged FcγRIIA (A-I) or FcγRIIB2 (J-R) were treated with (D-I, M-R) or without (A-C, J-L) agIgG for 80 minutes. Receptors were localized by immunofluorescence with anti-myc antibodies (A, D, G, J, M, P). Rhodamine-transferrin (B, E, K, N). AgIgG (H, Q). Merged images show receptors in green and transferrin (C, F, L, O) or agIgG (I, R) in magenta. White in merged images shows co-localizations of transferrin or agIgG with the receptors. Arrows in (F) highlight instances of lack of FcγRIIA co-localization with transferrin (Tf). Arrows in (R) highlight instances of agIgG adjacent to but not co-localizing with the internal FcγRIIB2 pool. Scale bar: 10μm. Images were analyzed by widefield microscopy. Representative of three experiments.

Rab11-GFP receptor agIgG receptor/Rab11-GFP

IIA 20’

ABCD

IIA 80’

EFGH

IIB2 20’

IJKL

IIB2 80’

MNOP

Fig. 3-7 FcγRIIB2, but not FcγRIIA is sorted into Rab11-positive recycling endosomes after internalization. ts20 cells stably expressing MH-tagged FcγRIIA (A-H) or FcγRIIB2 (I-P) were transiently transfected with Rab11-GFP. After treatment with agIgG for 20 minutes (A-D, I-L) or 80 minutes (E-H, M-P), receptors and agIgG were localized by immunofluorescence with anti-myc antibodies and anti-human antibody respectively. Rab11 (A, E, I, M). Receptors (B, F, J, N). agIgG (C, G, K, O). Merged pictures show Rab11 in green and receptors in red. Yellow in merged images and arrows in M,N,P indicate co- localization of Rab11 and FcγRIIB2. Scale bar: 10μm. Images were analyzed by widefield microscopy. Representative of two experiments.

3.3.3 Extracellular domains of FcγRIIA and FcγRIIB2 do not determine their differential trafficking

Although the extracellular domains of FcγRIIA and FcγRIIB2 are highly similar, the affinities of the His131 form of FcγRIIA for IgG1, IgG2, and IgG3 are several-fold higher than those of FcγRIIB2 (Bruhns et al., 2009; Maenaka et al., 2001). The divergent localization of

FcγRIIB2 and agIgG at later times after endocytosis (Fig. 3-6P-R) implies that the agIgG dissociates from the receptor after internalization. Thus, one possible determinant of the differential sorting of FcγRIIB2 and FcγRIIA could be a difference in release of ligand due to their differing extracellular domains. To address this possibility, chimeric receptors were generated in which the extracellular domain of FcγRIIA was fused to the transmembrane and cytoplasmic domains of FcγRIIB2 (“IIA-IIB2”) or vice versa (“IIB2-IIA”). Western blotting was performed at different time points after induction of agIgG endocytosis in cells expressing these receptors. As shown in Fig. 3-8, the chimeric receptor with FcγRIIA extracellular domain and FcγRIIB2 intracellular domain (IIA-IIB2) showed no obvious drop in receptor level after agIgG internalization, while conversely the chimeric receptor with FcγRIIB2 extracellular domain and FcγRIIA intracellular domain (IIB2-IIA) was degraded. Thus, the differential sorting of these receptors following endocytosis is presumably due to their divergent cytoplasmic domains.

3.3.4 Sorting of FcγRIIA and FcγRIIB2 upon persistent cross-linking with antibody complexes

While a difference in affinity for IgG apparently does not explain the difference between FcγRIIA and FcγRIIB2 sorting, the question nonetheless remains whether release from clustering ligand is necessary for sorting of the low affinity FcγRIIB2 away from a degradative

IIA IIB2 IIA IIB2

IIB 2 IIA IIB2 IIA

receptor: none IIA IIB2 IIA-IIB2 IIB2-IIA agIgG(min) 90 - 90 180 - 90 180 - 90 180 - 90 180

43 anti-myc

β-actin

l 140

e IIA v

120 IIB2 le

r IIA-IIB2 o

t 100 IIB2-IIA

p e

c 80

e r

l 60 ia

it 40 in

f 20 o

% 0 0 50 100 150 200 time (min)

Fig. 3-8 Extracellular domains of FcγRIIA and FcγRIIB2 do not determine their differential trafficking. ts20 cells were stably transfected with MH-tagged wild type FcγRIIA (IIA), FcγRIIB2 (IIB2) or chimeric receptors comprised of the extracellular domain of FcγRIIA fused to the transmembrane and cytoplasmic domains of FcγRIIB2 (IIA-IIB2) or vice versa (IIB2-IIA). agIgG was added to the cells and incubated at 34oC for indicated times. Lysates were analyzed by western blotting with anti-myc antibodies. The blot was stripped and reprobed with β-actin antibody. Brackets indicate the receptor positions. Representative of three experiments. The graph shows the quantification of receptor degradation after agIgG internalization. Y axis shows the percentage of receptor level after agIgG stimulation relative to the initial receptor level normalized to actin. Dash line with diamonds: IIA; solid line with squares: IIB; solid line with triangles: IIA-IIB2; dash line with crosses: IIB2-IIA. n = 3.

fate, since persistence of ligand binding is thought to be an important factor determining lysosomal sorting of receptors (Longva et al., 2002). To address this question, I attempted to induce a more persistent clustering of FcγRIIB2 by engaging it with anti-FcγRII antibody AT10 followed by cross-linking receptors with secondary antibody, rather than through the low affinity binding of multivalent agIgG. When FcγRIIB2-expressing cells were incubated with

AT10 alone (with no secondary antibody), the AT10 was delivered to transferrin-positive endosomes, suggesting that either there is constitutive cycling of FcγRIIB2 between cell surface and recycling endosomes in the unclustered state or that dimerization of receptors by bivalent antibody is sufficient to trigger such cycling (Fig. 3-9A-D). Upon clustering receptors with

AT10 and secondary antibody, in contrast to the release of ligand that occurs with agIgG,

AT10-antibody complexes remained co-localized with the receptors, presumably due to their binding through a higher affinity interaction. Moreover, both FcγRIIB2 and the AT10 complexes were directed to recycling endosomes (Fig. 3-9E-H). Consistent with this localization, neither FcγRIIB2 nor AT10 antibody complexes were degraded, in contrast to the case for FcγRIIA similarly engaged with AT10 and secondary antibody (Fig. 3-10). Thus, even under conditions of persistent oligomerization, FcγRIIB2 avoids a degradative fate.

Of note, when FcγRIIA-expressing cells were incubated with Fab fragments of the anti-

FcγRIIA antibody IV.3 and chased at 34oC, the Fab was internalized to transferrin-positive endosomes, suggesting that constitutive cycling of FcγRIIA can occur in the absence of clustering (Fig. 3-11B-D). In contrast, when whole IV.3 antibodies were used, FcγRIIA was sorted away from transferrin-positive recycling endosomes (Fig. 3-11E-H) and showed localization in LAMP1-positive lysosomes (Fig. 3-11I-K). FcγRIIA dimerization by bivalent antibodies (or possibly trimerization through additional engagement of the Fc region) appears to be sufficient to drive this sorting event. With whole IV.3 followed by cross-linking with

Tf FcγRIIB2 AT10 Tf/FcγRIIB2/AT10 RIIB2 γ AT10 Fc ABC D RIIB2 γ Fc AT10+x-link E F G H

Fig. 3-9 Sorting of FcγRIIB2 after engagement with anti-FcγRII antibodies. ts20 cells expressing MH-tagged FcγRIIB2 were incubated at 4°C with AT10. Cells were then washed and incubated at 4°C with cy5-anti-mouse secondary antibodies (E-H) to cross-link receptors or had no secondary antibody treatment (A-D). Cells were then chased at 34oC for 60 minutes where they were immediately fixed. FcγRIIB2 was localized by immunofluorescence with a polyclonal rabbit antibody directed against its intracellular domain (B, F). Cy5-labeled prebound secondary antibodies were detected in (G). Uncrosslinked AT10 (C) were detected by immunofluorescence with anti-mouse secondary antibodies after fixation. Alexa488-transferrin (A, E). Merge images (D, H). Images were analyzed by widefield microscopy. Representative of three experiments.

Fig. 3-10 FcγRIIA, but not FcγRIIB2, undergoes degradation after cross-linking via anti- FcγRII antibody complexes. ts20 cells expressing FcγRIIA-MH (A) or FcγRIIB2-MH (B) or untransfected cells were stimulated with agIgG or AT10 antibodies with secondary antibody cross-linking for indicated times. Cell lysates were analyzed by western blotting with anti-myc antibodies (A, B) or directly with HRP-conjugated anti-mouse antibody to detect heavy chains of AT10 antibody (C). Blots were stripped and reprobed with β-actin antibodies. Representative of two experiments.

IV.3 Fab (4 C) IV.3 Fab Tf Tf/IV.3 Fab RIIA γ Fc IV.3 Fab IV.3 ABCD TfFcγRIIA IV.3 Tf/FcγRIIA/IV.3 RIIA γ IV.3 Fc

E F G H LAMP1 IV.3 IV.3/LAMP1 RIIA γ IV.3 Fc IJK RIIA γ Fc IV.3+x-link LMN

Fig. 3-11 Sorting of FcγRIIA after engagement with anti-FcγRIIA antibodies. ts20 cells expressing MH-tagged FcγRIIA were incubated at 4°C with IV.3 Fab (A-D) or intact IV.3 (E- N). Cells were then washed and incubated at 4°C with IgG2b-specific cy5-anti-mouse secondary antibodies (L-N) to cross-link receptors, with cy5-labelled Fab fragments of anti- mouse secondary antibody to label IV.3 Fab (A-D), or had no secondary antibody treatment (E- K). Cells were then chased at 34oC for 60 minutes, except in A, where they were immediately fixed. FcγRIIA was localized by immunofluorescence with anti-myc (F). Cy5-labeled prebound secondary antibodies were detected in (A, B, M). Uncrosslinked IV.3 (G, J) were detected by immunofluorescence with anti-mouse secondary antibodies after fixation. Alexa488-transferrin (C, E). LAMP-1 (I, L). Merge images (D, H, K, N). Isotype-specific anti-mouse secondary antibodies were used for immunofluorescence in F-N (anti-myc and anti- LAMP1 are both IgG1 antibodies; IV.3 is an IgG2b). Yellow in merged images indicates co- localizations. Images were analyzed by widefield microscopy. Representative of three experiments. 65

secondary antibody, robust delivery of FcγRIIA to lysosomes was seen (Fig. 3-11L-N).

3.3.5 FcγRIIA dimerization induces receptor tyrosine phosphorylation

FcγRIIA cross-linking by immune complexes on the cell surface is required for ITAM- mediated receptor tyrosine phosphorylation which triggers downstream pro- inflammatory signalling. Dimerization of FcγRIIA with bivalent whole IV.3 antibodies is sufficient to drive

FcγRIIA endocytosis and subsequent sorting to lysosomes (Fig. 3-11). Here, I examined whether FcγRIIA phosphorylation can be initiated by such receptor dimerization. FcγRIIA- expressing ts20 cells were incubated on ice with IV.3 Fab fragments, bivalent IV.3 whole antibody or IV.3 antibody complexes before incubation in warm medium to trigger endocytosis.

Extensive clustering of FcγRIIA with IV.3 complexes triggers receptor phosphorylation whereas IV.3 Fab binding to FcγRIIA does not (Fig. 3-12). FcγRIIA dimerization by whole

IV.3 antibodies is sufficient to trigger FcγRIIA phosphorylation. FcγRIIA phosphorylation is maximal even upon engagement on ice and is sustained for 10 minutes.

3.3.6 Sorting of FcγRIIA and FcγRIIB2 in primary human myeloid cells

Next, I attempted to recapitulate these findings in primary human myeloid cells, including monocytes, monocyte-derived macrophages and immature monocyte-derived DCs

(moDCs). Monocytes were isolated from peripheral blood of healthy donors and differentiated ex vivo into macrophages or moDCs by cytokine treatments. Monocytes express a very low level of FcγRIIB which can be upregulated by overnight treatment with IL-4 and IL-10 (Joshi et al., 2006). It has been noted by Boruchov et al that FcγRIIA and FcγRIIB2 are the only FcγRs that moDCs express (Boruchov et al., 2005). To assess the surface expression of FcγRIIA and

FcγRIIB2, these myeloid cells were incubated on ice with IV.3 antibody, which preferentially

Receptor none FcγRIIA Stimulus IV.3+x-link - IV.3 Fab IV.3 IV.3+x-link time (min) - - 05100510 0510 58

4G10 (pTyr)

anti-myc (receptor) Fig. 3-12 ITAM-mediated tyrosine phosphorylation of FcγRIIA with different degrees of cross-linking. ts20 cells expressing FcγRIIA-MH or untransfected cells were incubated on ice with IV.3 Fab fragments, IV.3 whole antibody or IV.3 with secondary antibody cross-linking. This is followed by 5 minutes or 10 minutes chase at 34oC or immediate lysis of cells (0 min). Cell lysates were immunoprecipitated with anti-myc antibody A14 and analyzed by western blotting with anti-phosphotyrosine antibody 4G10. The blot was stripped and reprobed with anti-myc antibody 9E10. Representative of five experiments.

recognizes FcγRIIA, or 4F5 antibody, which preferentially recognizes the extracellular domain of FcγRIIB, before fixation and analysis by microscopy. Table 3-1 summarizes the FcγRIIA and FcγRIIB levels I observed in various human myeloid cells.

Table 3-1: the expression of FcγRIIA and FcγRIIB in various human myeloid cells

cell Dextran Cell type FcγRIIA level FcγRIIB level size uptake

high but heterogeneous a fraction of cells low and monocytea small expression of have high FcγRIIB heterogeneous FcγRIIA in most expression cells

All cells have very high expression of high but low FcγRIIB level by macrophageb Large FcγRIIA in most heterogeneous day 6 of cell culturing cells and drop over time.

A large fraction of A small fraction of immature large high cells express high cells express low moDC c level of FcγRIIA level of FcγRIIB a: n = 3 b: n = 5 c: n = 3

As documented by other groups (Guriec et al., 2006; Liu et al., 2006; Su et al., 2007),

FcγRIIA is expressed on different human myeloid cells at a level sufficient for clear visualization of delivery of FcγRIIA-specific antibody to endosomes when internalized (Table

3-1). In contrast, there seems to be variations in FcγRIIB expression among donors as previously described (Boruchov et al., 2005; Su et al., 2007). There are large cell-to-cell heterogeneities in the surface level of FcγRIIB on monocytes and moDCs. The fraction of

FcγRIIB-expressing cells is relatively small (<40%) and the level of FcγRIIB in these cells is relatively low. Furthermore, the surface expression of FcγRIIB in monocyte-derived macrophages is very low and can hardly be detected by microscopy.

In monocyte-derived macrophages, FcγRIIA was engaged with IV.3 with or without secondary antibody cross-linking. Cells were preloaded with fluorescent dextran to label the lysosomal compartment. At early times after engagement, IV.3 or IV.3 complexes were internalized into peripheral early endosomes that were positive for EEA1 (Fig. 3-13) and negative for dextran (Fig. 3-14A-C, G-I). By 60 minutes post-internalization, both IV.3 alone and IV.3-secondary antibody complexes moved into dextran-positive central lysosomes (Fig. 3-

14D-F, J-L). The trafficking of IV.3 antibody complexes internalized by FcγRIIA was also analyzed in human monocytes and immature monocyte-derived DCs (moDC). IV.3 complexes were taken up into dextran-negative punctate endosomal compartments as early as 5 minutes after receptor clustering (Fig. 3-15A-C, G-I). FcγRIIA-mediated internalization of IV.3 complexes from the cell surface was also detected in immature moDC by flow cytometry (Fig.

3-16). These antibody complexes are translocated into dextran positive lysosomes 60 minutes after FcγRIIA engagement (Fig. 3-15D-F, J-L).

3.3.7 FcγRIIA sorting for degradation does not require Src family kinase activity (SFK) or receptor ubiquitylation at lysine residues

Our results with chimeric receptors indicate that it is most likely the divergentintracellular domains of FcγRIIA and FcγRIIB2 that dictate their differential sorting. The intracellular domain of FcγRIIA contains an ITAM that is phosphorylated by SFK to induce downstream signalling cascades (Daeron, 1997). The Booth lab has previously reported that FcγRIIA engagement in transfected ts20 cells leads to receptor phosphorylation, and that both phosphorylation of FcγRIIA and phagocytosis driven by this receptor are inhibited by the SFK inhibitor PP1 (Mero et al., 2006). Here, I tested the effect of PP1, a SFK inhibitor, on FcγRIIA intracellular sorting. While degradation was blocked by bafilomycin treatment, PP1 did not

EEA1 IV.3 Ab complex (10’) EEA1/IV.3 Ab complex

N=1 100107

10uM

C AB

Fig. 3-13 Antibody complexes internalized by FcγRIIA are first sorted into EEA1+ early endosomes in human macrophages. Monocyte-derived human macrophages purified by adherence for 6 days of differentiation were stimulated at 4oC with IV.3 followed by cross- linking with Alexa488-labeled anti-mouse secondary antibody. Cells were then chased at 37oC for 10 minutes. The co-localization of IV.3 antibody complexes (A) with EEA1+ early endosomal compartments (B) were analyzed by immunofluorescence with anti-EEA1 antibodies. Merged images show IV.3 antibody complexes in green and EEA1 in magenta (C). Arrows highlight incidences of co-localization of EEA1 and IV.3 antibody complexes. Scale bar: 10μm. Preliminary experiment. Images were analyzed by widefield microscopy.

dextran IV.3 dextran/IV.3 IV.3 10 min AB C IV.3 60 min DE F k .3+x-lin 10 min V I GH I k .3+x-lin 60 min V I JK L

Fig. 3-14 Antibody complexes internalized by FcγRIIA are sorted to lysosomal compartments in human macrophages. Monocyte-derived human macrophages were preloaded with Alexa647-conjugated dextran. Cells were then incubated at 4oC with Alexa488- conjugated IV.3 (A-F) or unlabeled IV.3 followed by Alexa488-anti-mouse secondary antibody (G-L). Cells were then chased at 37oC for 10 minutes (A-C, G-I) or 60 minutes (D-F, J-L). Merged images show dextran in red and IV.3 in green (C, F, I, L). Yellow in merged images indicates co-localization of dextran and IV.3. Images were analyzed by confocal scanning microscope. Representative of three experiments.

dextran IV.3 Ab complex dextran/IV.3 5 min monocyte

ABC 60 min monocyte

DEF re moDC u 5 min

immat GHI re moDC u 60 min

immat JK L

Fig. 3-15 The sorting of antibody complexes internalized by FcγRIIA to lysosomes in human monocytes and immature moDC. Day 2 isolated monocytes (A-F) or day 6 differentiated immature moDCs (G-N) were preloaded with Alexa647-conjugated dextran overnight or for one hour respectively. After incubation at 4oC with IV.3 and Alexa488-labeled anti-mouse secondary antibody (B, E, H, K), cells were chased at 37oC for 5 minutes (A-C, G-I) or 60 minutes (D-F, J-L). Dextran (A, D, G, J). Merged images showdextran in red and IV.3 in green (C, F, I, L). Yellow in merged images indicates co-localization of dextran and IV.3. Images were analyzed by confocal scanning microscope. Representative of two experiments.

4 10 100 71.7 28.3 100 20 min 0 min 3 20 min 10 80 80 no stain .3 0 min

V no stain 60 I 60 2 d 10

FL4-H n %ofMax %ofMax 40

u 20 min 40 o 1 0 min b 10 - 20

no stain 20 e

c C

a A B 0 f 0 10 0

r 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 10 10 10 10 10 10 10 FL10 1-H 10 10 10 10 10 10 10 u FL4-H

s FL1-H total IV.3 total IV.3 surface-bound IV.3 Fig. 3-16 FcγRIIA-mediated internalization of anti-FcγRIIA antibodies in immature moDC. Day 6 differentiated moDCs were incubated with Alexa488-IV.3 (total IV.3 level) on ice for 20 minutes. This is followed with (red line) or without (blue line) 20 minutes chase at 37oC to allow for endocytosis to start. Cells were then stained with cy5-anti-mouse antibody on ice for 10min to assess the level of surface-bound IV.3 before being fixed and analyzed by flow cytometry. The level of surface-bound IV.3 (A, C) and total IV.3 (A, B) were shown. n=4

prevent agIgG-induced FcγRIIA degradation (Fig. 3-17A, B) or slow down its rate (Fig. 3-18A).

However, PP1 abolished the phagocytosis of sheep red blood cells by FcγRIIA (Fig. 3-19A, B), confirming the efficacy of the inhibitor. These findings indicate that SFK-mediated receptor tyrosine phosphorylation is not essential to direct FcγRIIA to lysosomes during FcγRIIA- mediated endocytosis. Consistent with the observed lack of effect of PP1 on FcγRIIA degradation, agIgG and FcγRIIA are still sorted into a punctate compartment in the presence of

PP1 (Fig 3-17C, D). I also tested the involvement of phosphatidylinositol 3-kinases (PI3K) in

FcγRIIA sorting. PI3K has been shown to be involved in the accumulation of phosphotidylinositol 3-phosphate (PI3P) at the membrane of endosomal, phagosomal or vacuolar structures, important for their maturation (Scott et al., 2002). No inhibition of

FcγRIIA degradation was observed when cells were pre-treated with PI3K inhibitor LY294002

(Fig. 3-17A, Fig. 3-18B) whereas both FcγRIIA-mediated phagocytosis and accumulation of phosphotidylinositol 3-phosphate on vacular membranes were inhibited by LY294002 (Fig. 3-

19A, C, D-G). This suggests that PI3K does not play an important role in lysosomal targeting of FcγRIIA. Next, I examined the requirements of SFK for FcγRIIA degradation in THP-1 cells. PP1 partially inhibits but does not abolish FcγRIIA degradation (Fig. 3-20).

Ubiquitylation of surface receptors can function both to trigger endocytosis and as a sorting signal driving their delivery to lysosomes (Katzmann et al., 2002; Stang et al., 2004;

Staub et al., 2006). FcγRIIA is ubiquitylated upon engagement by immune complexes and an active ubiquitylation machinery is required for receptor endocytosis (Booth et al., 2002; Mero et al., 2006). This raises the possibility that receptor ubiquitylation may also account for its lysosomal sorting. The intracellular domain of FcγRIIA contains five lysine residues that can serve as potential ubiquitylation sites. I assessed the degradation of a mutated version of

FcγRIIA in which all five lysine residues were mutated to arginine (5KR). ts20 cells

A receptor none FcγRIIA inhibitor ---PP1 LY BAF agIgG +-+++ + 55

anti-myc (FcγRIIA)

β-actin total receptor level (anti-myc)

B 120 I

F 100 +PP1 -PP1

l M l 80 ia

it 60 in

f 40 o

% 20

0 0 180 time (min) -PP1 +PP1

C D

receptor/agIgG

Fig. 3-17 Src family kinase (SFK) activity is not required for FcγRIIA sorting for degradation. (A) ts20 cells or ts20 cells expressing MH-tagged FcγRIIA were stimulated with or without agIgG for 4 hours in the presence of PP1, LY294002 (LY) or bafilomycin as indicated. Cell lysates were prepared and subjected to western blotting with anti-myc antibody 9E10. The blot was stripped and reprobed with anti-β-actin antibody. Representative of five experiments. (B) Cells were stimulated with agIgG for 180 minutes with or without PP1 treatment and receptors remaining were measured by intracellular staining with anti-myc antibody and flow cytometry. The total receptor level is expressed as a percentage of the initial mean fluorescence intensity at time 0 after background subtraction of fluorescence intensity of untransfected ts20 cells stained with anti-myc. Error bars indicate S.D. n =3. (C, D) FcγRIIA-MH expressing ts20 cells were pretreated with (D) or without (C) PP1 before stimulation with agIgG for 80 minutes. Receptors and agIgG were then localized by immunofluorescence with anti-myc or anti-human antibodies respectively. Merged images show agIgG in green and FcγRIIA in magenta. White in merged images indicates co- localizations of agIgG and receptors. Arrows highlight incidences of co-localization. Scale bar: 20 μm. Images were analyzed by widefield microscopy. Representative of four experiments.

A receptor: none IIA-MH PP1 - - + agIgG(min) 60 - 60 90 120 150 180 - 60 90 120 150 180 55

anti-myc (IIA)

β -actin

120 l

e -PP1 v 100

le +PP1

r o

t 80

e c

e 60

r l

ia 40 it

in 20

f o

0 % 0 50 100 150 200 time (min)

B agIgG(min) - 240 90 120 150 180 inhibitor - BFM - LY - LY - LY - LY

55 anti-myc (IIA)

β -actin

Fig. 3-18 Effect of PP1 and LY294002 on the kinetics of FcγRIIA degradation. ts20 cells expressing MH-tagged FcγRIIA were pretreated with or without PP1 (A) or LY294002 (LY) (B) 30 minutes before stimulation with agIgG. Cells were then incubated at 34oC for different lengths of time. FcγRIIA level after 240 minutes of agIgG stimulation in the presence of bafilomycin was also analyzed. Cell lysates were prepared and subjected to western blotting with anti-myc antibody. The blot was stripped and reprobed with anti-β-actin antibody. (A) representative of three experiments. The graph shows the quantification of FcγRIIA degradation after agIgG internalization in the presence and absence of PP1. Y axis shows the percentage of FcγRIIA level after agIgG stimulation relative to the initial FcγRIIA level normalized to actin. n = 3. (B) preliminary experiment.

control +PP1 +LY294002

A B C non-internalized SRBCs phagocytosed SRBCs

control 1hr LY294002

DE 2hr LY294002 3hr LY294002

FG Fig 3-19 Efficacy test of PP1 and LY294002. (A-C) ts20 cells stably expressing MH-tagged FcγRIIA were incubated with rabbit IgG opsonised sheep red blood cells (SRBCs) for 40 minutes after pre-treating cells with PP1 (B) or LY294002 (C) for 4 hours or without pre- treatment (A). SRBCs remaining outside (green) and total SRBC outside and inside the cells (red) were detected with fluorophore-conjugated secondary anti-rabbit antibodies. Yellow indicates non-internalized SRBCs whereas red shows phagocytosed SRBCs. n= 2. (D-G) ts20 cells were transiently transfected with 2-FYVE-GFP (two tandem FYVE domains attached to GFP). After treating cells with LY294002 for 1 hour (E), 2 hours (F), 3 hours (G) or without treatment, samples were fixed and analyzed by Zeiss Axiovert 200M microscope. Arrow indicates the accumulation of 2-FYVE-GFP on the vacuolar membrane. Images were analyzed by widefield microscopy. Preliminary experiment.

IP IV.3 - + inhibitors - ---BAFBAF PP1 PP1 agIgG - - 90’ 180’ 90’ 180’ 90’ 180’ 43

W estern Blotting quantification 120

l -PP1 e P=0.0002 v 100

+PP1 le

A 80 I P=0.01

l I l 60

ia it

in f

o 20 % 0 0180 time (min) Fig. 3-20 Effect of PP1 on agIgG-induced FcγRIIA degradation in THP-1. agIgG was added to THP-1 cells and incubated for different lengths of time at 37oC following pre- treatment with bafilomycin (lane 5, 6) or PP1 (lane 7, 8) or no inhibitor (lane 1-4). FcγRIIA was immunoprecipitated with mouse IV.3 antibody against extracellular domain of FcγRIIA, followed by western blotting with rabbit anti-FcγRIIA intracellular antibody. The blot is representative of three experiments. The bar graph shows the quantification of FcγRIIA degradation after three hours of agIgG internalization in the presence and absence PP1 (lane 2, 4, 8 of the blot). Y axis shows the percentage of FcγRIIA level after agIgG stimulation relative to the initial FcγRIIA level normalized to the light chain of immunoprecipitation antibody IV.3 (the recovery control). n = 3.

stably expressing either wild type FcγRIIA or 5KR (with 5KR level lower than wild type

FcγRIIA) were treated with IV.3 and cross-linking secondary antibody to trigger endocytosis.

Antibody complexes internalized by either wild-type FcγRIIA or IIA-5KR were both delivered to LAMP1-positive lysosomes (Fig. 3-21A-F). Moreover, the lack of lysines does not prevent

FcγRIIA degradation (Fig. 3-21G), indicating that direct ubiquitylation of lysines in FcγRIIA is not essential for its degradation, and suggesting that non-lysine residues or other sorting signals in the cytoplasmic domain of FcγRIIA may be involved in this step of its trafficking.

3.3.8 FcγRIIA and FcγRIIB2 trafficking upon co-engagement

Our heterologous transfection model has the advantage of allowing the analysis of the intracellular trafficking capabilities of FcγRIIA and FcγRIIB2 in isolation. However, in cells co-expressing FcγRIIA and FcγRIIB2, both receptors would be expected to be simultaneously co-engaged by multivalent immune complexes. If FcγRIIA and FcγRIIB2 carry different sorting signals, what is the result of such receptor co-engagement? On one hand, FcγRIIA might have a dominant effect, pulling FcγRIIB2 towards lysosomal degradation. Conversely, co-engaged FcγRIIB2 might reroute FcγRIIA away from such a fate. A third possibility is that dissociation of immune complexes from the receptors allows them to sort to their respective fates independently of each other.

To address this question, ts20 cells stably expressing MH-tagged FcγRIIA were transiently transfected with untagged FcγRIIB2. AgIgG was added to these cells to co-engage

FcγRIIA and FcγRIIB2. The subcellular localization of FcγRIIA, FcγRIIB2 and agIgG were determined by immunofluorescence (Fig. 3-22A-H). After 10 minutes of agIgG stimulation,

FcγRIIA and FcγRIIB2 both co-localized with agIgG in dispersed peripheral endosomes (Fig.

3-22A-D). However, by 90 minutes after agIgG stimulation, FcγRIIA and agIgG were

LAMP1 IV.3 LAMP1/IV.3 IIAwt

ABA B C 5KR

D E F G receptor none IIA-wt 5KR IV.3+2ary: - - 090 180 0 90 180 (min)

IP: IV.3 WB: anti-FcγRIIA

Fig. 3-21 Receptor lysine residues are not necessary for FcγRIIA degradation. (A-F) ts20 cells expressing MH-tagged wild type FcγRIIA (IIAwt: A-C) or His-tagged FcγRIIA with all five intracellular lysines mutated to arginine (5KR: D-F) were incubated with IV.3 and IgG2b- specific anti-mouse secondary antibody at 4oC and then chased at 34oC for 80 minutes. IV.3 antibody complexes (B,E). LAMP1 (A, D). Merged images show LAMP1 in magenta and IV.3 in green (C,F). White in merged images denotes co-localizations of LAMP1 and agIgG. Arrows highlight co-localization. Images were analyzed by widefield microscopy. Representative of three experiments. (G) ts20 cells expressing IIAwt or 5KR or untransfected cells were incubated with or without IV.3 antibody complexes at 4oC, followed by chase at 34oC for indicated times. Following lysis of the cells, the receptors were immunoprecipitated with IV.3 and blotted with rabbit antibody against the intracellular domain of FcγRIIA. Bracket indicates position of FcγRIIA. Arrow indicates non-specific cross-reacting band. Representative of five experiments.

Fig. 3-22 FcγRIIA and FcγRIIB2 sorting upon co-engagement with agIgG. (A-J) ts20 cells stably expressing FcγRIIA-MH (IIA-MH) were transiently transfected with untagged FcγRIIB2. The cells were incubated with agIgG on ice for 20 minutes, then chased at 34oC for 10 minutes (A-D) or 90 minutes (E-H). AgIgG, FcγRIIA, and FcγRIIB2 were detected by immunufluorescence using anti-human (A, E), anti-myc (B, F), and rabbit anti-FcγRIIB2 (C, G) antibodies, respectively. Merged images show FcγRIIA-MH in green and FcγRIIB2 in magenta (D, H). White in merged images indicates co-localizations of FcγRIIA-MH and FcγRIIB2. Representative of three experiments. (I) ts20 cells stably expressing FcγRIIA-MH were transiently co-transfected with untagged FcγRIIB2 and GFP. FcγRIIA-MH levels before and after 3 hours incubation with agIgG were analyzed by intracellular flow cytometry with anti-myc. Right panel shows staining of untransfected ts20 cells. (J) ts20 cells stably expressing FcγRIIB2-MH were transiently co-transfected with untagged FcγRIIA and GFP. FcγRIIB2-MH levels before and after 3 hour incubation with agIgG were analyzed by intracellular flow cytometry with anti-myc. Right panel shows staining of untransfected ts20 cells. The red lines are added to facilitate comparison of receptor levels before and after agIgG treatment. Images were analyzed by widefield microscopy. Representative of three experiments.

colocalized in central puncta while FcγRIIB2 showed a distinct localization in a diffuse central, probably recycling endosomal pool, as was seen with individually expressed receptors (Fig. 3-

22E-H). As an alternative approach, the effect of co-engagement on receptor degradation was assessed by transiently expressing FcγRIIB2 in cells that stably express FcγRIIA, or vice versa, and measuring degradation of the stably expressed receptor by flow cytometry (Fig. 3-22I,J).

Expression of cotransfected GFP was used as a measure of transient receptor expression (Fig.

3-22I, J and Fig. 3-23). FcγRIIA was still degraded in cells expressing a range of levels of

FcγRIIB2 (Fig. 3-22I), and conversely there was no obvious degradation of FcγRIIB2 in cells expressing a range of levels of FcγRIIA (Fig. 3-22J). Thus, our results suggest that dissociation of FcγRII from immune complexes after internalization allows independent sorting of FcγRIIA and FcγRIIB2 receptors to distinct final fates.

As shown in Fig 3-9, antibody complexes which bind to FcγRIIB through a higher affinity interaction than agIgG are transported together with the receptors to their destined fates instead of dissociating from the receptors before sorting occurs. Next, I sought to investigate if persistent co-clustering of FcγRIIA and FcγRIIB2 with AT10 antibody complexes alters their sorting patterns. In ts20 cells co-expressing both receptors, FcγRIIA, FcγRIIB2 and antibody complexes are sorted into dispersed punctate endosomes 10 minutes after co-engagement (Fig.

3-24A-D). After 90 minutes of receptor co-clustering, FcγRIIA is delivered to central puncta, probably lysosomes, containing clustering antibody complexes, as was observed upon agIgG co-engagement (Fig. 3-24E, F). The majority of FcγRIIB2 is still found in diffuse central endosomes, some co-localization with FcγRIIA in central puncta is also seen (Fig. 3-24E, H).

This finding suggests that FcγRIIB2 can be rerouted from recycling pathway to a degradative fate to some extent upon persistent co-engagement with FcγRIIA.

Fig. 3-23 Expression of cotransfected GFP as a measure of transient receptor expression. GFP expression correlates with expression of transiently transfected receptors. (A) Untransfected ts20 cells (no receptors or GFP). (B-C) ts20 cells stably expressing FcγRIIA-MH with (C) or without (B) transient cotransfection with FcγRIIB2-MH and GFP. Total Myc staining is higher in GFP-high cells, reflecting transient expression of the tagged FcγRIIB2-MH in addition to the stable FcγRIIA-MH. Note that, due to the limitations of transfection efficiency, the major population in the transient transfectants represents non-transfected cells, expressing no additional FcγRIIB2-MH. The fluorescence in the GFP channel of these cells was routinely observed to be higher than that of cells that were not subject to transfection at all (B), apparently due to contamination of the untransfected population with some GFP from the GFP-expressing cells during culture or after detachment of the cells. Transient receptor expression correlates with GFP fluorescence above this baseline (C). Preliminary experiment.

AT10 Ab complex IIA-MH IIB2 IIA-MH/IIB2

10’

AB CD

90’

E F G H

Fig. 3-24 FcγRIIA and FcγRIIB2 sorting upon co-engagement with anti-FcγRII antibody. ts20 cells stably expressing FcγRIIA-MH (IIA-MH) were transiently transfected with untagged FcγRIIB2. The cells were incubated with AT10 followed by Alexa488 anti-mouse antibodies each on ice for 20 minutes, then chased at 34oC for 10 minutes (A-D) or 90 minutes (E-H). FcγRIIA-MH, and FcγRIIB2 were detected by immunufluorescence using goat anti-IIA (B,F) and rabbit anti-FcγRIIB2 (C,G) antibodies, respectively. AT10 antibody complexes (A,E). Merged images shows FcγRIIA-MH in red and FcγRIIB2 in green (D, H). Yellow in merged images denotes co-localizations of FcγRIIA-MH and FcγRIIB2. Arrows highlight co- localization. Images were analyzed by widefield microscopy. Representative of three experiments.

3.4 Discussion

In this study, I have directly compared the intracellular trafficking of FcγRIIA and

FcγRIIB2. My results demonstrate that in addition to their opposing signaling functions, they have very different trafficking behaviours. Degradation of the activating FcγRIIA after its internalization may serve as a mechanism for ensuring appropriate termination of inflammatory signaling after initial encounter with immune complexes. In contrast, persistence of FcγRIIB2 may help to maintain the ability of myeloid cells to continue clearing immune complexes.

When analyzed by western blotting, both FcγRIIA and FcγRIIB2 appear as doublets (Fig.

3-2A, 3-8, 3-18) or in some cases smears (Fig. 3-4A, 3-12, 3-21G) which may reflect different glycosylation of the receptors. Notably, the rates of degradation of the two FcγRIIA bands seem different as the higher molecular weight receptor was degraded faster than the ones at a lower molecular weight (Fig. 3-8, 3-18B). It is possible that the fast-moving band of the

FcγRIIA doublet in western blotting represents the nascent receptors in the endoplasmic reticulum to which immune complexes cannot gain access whereas the slow-moving band indicates the mature glycosylated receptors on the cell surface that interact with ligands.

Despite the slower rate of degradation of the faster-moving band, the level of this pool of

FcγRIIA still drops over time (Fig. 3-2, 3-18B), possibly due to movement of nascent receptors to the cell surface over time. To test this hypothesis, endoglycosidases which remove oligosaccharides from protein could be used prior to detection of FcγRIIA degradation by immunoblotting.

Incubation of FcγRIIA-expressing cells with Fab fragments of anti-FcγRIIA antibody IV.3 led to labelling of a recycling endosomal pool. This suggests that FcγRIIA, like human FcγRI

(Harrison et al., 1994), has the capacity to cycle between the cell surface and endosomal compartments in the absence of receptor clustering. Notably, I found that engagement of

FcγRIIA with bivalent whole antibodies was sufficient to induce not only FcγRIIA sorting to lysosomes but also its phosphorylation. It would be interesting to investigate how FcγRIIA dimerization may affect the magnitude and duration of FcγRIIA signalling.

My experiments with FcγRIIB2 highlight the fact that the receptor can dissociate from multivalent agIgG complexes after internalization. On first thought, one might expect that the interaction of receptors with multivalent complexes would be highly stable. Indeed, immune complexes can bind strongly to the cell surface due to the simultaneous interactions of multiple

Fc domains with multiple FcγRIIB2 (i.e. avidity) even though the strength of each individual interaction is weak. On the cell surface where receptor pool is continuously replenished by proteins from the intracellular storage, each Fc fraction in the immune complexes that becomes dissociated from FcγRIIB2 can be rebound to another unoccupied receptor. However, once the receptor-ligand complexes are delivered to endosomal compartments, individual FcγRIIB2 that dissociates from the internalized immune complexes is quickly sorted away from the sorting endosomes into the recycling pathway. This leads to a gradual decrease in the overall avidity of receptor-ligand complexes. In this context the multivalent nature of the complex may primarily have the effect of increasing the local concentration of IgG. The low affinity of the FcγRII thus will serve to facilitate the release of receptors from internalized immune complexes, allowing their spatial segregation from the complexes through endosomal fission events. My observations suggest that following co-engagement of FcγRIIA and FcγRIIB2 these two receptors are sorted independently, implying that the dissociation of receptors from agIgG can occur before committed sorting events. The low affinity of the FcγRII may therefore confer two distinct evolutionary advantages. As is often noted, the low affinity of FcγRs allows cells to respond specifically to multivalent immune complexes, rather than being permanently occupied by monomeric IgG present in serum. A second advantage, however, may be that low

affinity allows FcγRs to access distinct intracellular compartments even after co-engagement by immune complexes. The more persistent co-clustering of FcγRIIA and FcγRIIB2 with AT10 antibody complexes does not obviously redirect FcγRIIA sorting to the recycling pathway. A portion but not all of FcγRIIB2 was rerouted from the recycling pathway to lysosomal compartments upon co-engagement with FcγRIIA. These finding suggests that the lysosomal targeting of FcγRIIA is a more dominant sorting signal than recycling of FcγRIIB2 when co- engaged at higher affinity.

As for the effect of affinity not on the fate of the receptors but rather on that of internalized immune complexes, my findings with anti-FcγRII antibody imply that the release of immune complexes internalized via FcγRIIB from receptors is required for the immune complexes to undergo lysosomal degradation. The low affinity of human FcγRIIB for IgG would facilitate this release. It is noteworthy that among the murine low affinity FcγRs, the inhibitory receptor FcγRIIB binds to mouse IgG1 with higher affinity than the activating receptors FcγRIII and FcγRIV, and binds to IgG2a and IgG2b with a comparable affinity to

FcγRIII (Nimmerjahn et al., 2005). In contrast, among the human low affinity FcγR, FcγRIIB has a substantially lower affinity than the activating receptors FcγRIIA and FcγRIII for all human IgG isotypes except IgG4 (for which affinities are similarly low for all receptors)

(Bruhns et al., 2009). FcγR-mediated uptake of antigen in immune complexes by antigen presenting cells can lead to greatly increased efficiency of subsequent antigen presentation to T cells (Regnault et al., 1999). However, the consequences of antigen uptake via FcγRIIB in particular are unclear. In some studies murine FcγRIIB has been shown to be capable of facilitating antigen presentation (Amigorena et al., 1992; Antoniou and Watts, 2002; de Jong et al., 2006; Yada et al., 2003), while in others it has been seen to have an inhibitory effect

(Bergtold et al., 2005; Desai et al., 2007). One of the means by which FcγRIIB can impair

antigen presentation is by suppressing ITAM-induced DC maturation (Kalergis and Ravetch,

2002). In addition, however, FcγRIIB in mouse DCs can recycle bound immune complexes to the cell surface and prevent their delivery to degradative compartments that is required for antigen processing (Bergtold et al., 2005; Desai et al., 2007). Recycling of a portion of internalized immune complexes to the cell surface was also observed with rat FcγRIIB in liver sinusoidal endothelial cells (Mousavi et al., 2007). Such routing of immune complexes away from a degradative fate will depend on the persistence of their binding to receptors. If immune complexes are released from FcγRIIB after internalization in human antigen presenting cells, with delivery of the released immune complexes to lysosomes, the uptake via FcγRIIB would be expected to facilitate antigen processing. The pH sensitivity of receptor binding to immune complexes may also be an important factor determining their release in endosomal compartments, and it would be interesting to determine the extent to which this varies among different Fc receptors.

Studies of FcγRIIA in neutrophils have shown an extremely rapid degradation of the receptor occurring in two minutes following its ligation. This degradation appears to involve the action of the proteasome (Marois et al., 2009). My results with monocytic THP-1 cells indicate that, as in the transfected cell model, degradation of FcγRIIA occurs in lysosomes over the longer time frames typical for such degradation. Moreover, trafficking of anti-FcγRIIA antibodies to lysosomes was observed in monocyte-derived macrophages. These results suggest that while loss of FcγRIIA after its ligation is a common theme in both monocytic cells and neutrophils, the very rapid degradation of FcγRIIA seen in neutrophils may be a mechanistically unique feature of these cells. Another recent study concluded that FcγRI, but not FcγRIIA, can be delivered to lysosomes in monocytes within 10 minutes of receptor

engagement (Dai et al., 2009). My results indicate that FcγRIIA can also traffic to lysosomes, with the slower kinetics more typical of lysosomal sorting of surface receptors.

While the ITAM of the common Fc receptor-γ chain that associates with FcγRI is important for triggering downstream signalling upon stimulation of cells via this receptor, the cytoplasmic tail of FcγRI itself also has functional effects, controlling ligand binding and endocytosis through interactions with periplakin (Beekman et al., 2004). My finding that neither SFK nor PI3K is essential for FcγRIIA degradation suggests that the cytoplasmic tail of this receptor may also interacts with as yet unidentified cytosolic proteins distinct from the

ITAM signalling pathway. The SFK inhibitor PP1 has some inhibitory effects but does not completely block FcγRIIA degradation in THP-1 cells. This suggests that there may be multiple FcγRIIA-sorting mechanisms, including both SFK-dependent and SFK-independent pathways. This difference between ts20 transfected cell model and THP-1 may be due to cell type differences such as the lack of key protein modulators downstream of FcγRIIA in ts20 cells. The lack of requirement for lysine residues also argues against a requirement for direct ubiquitylation at lysine residues of the receptor for its degradation, though we cannot exclude a role for non-conventional ubiquitylation of nonlysine residues (Wang et al., 2007), or ubiquitylation of a receptor-associated protein.

It is also possible that receptor sorting is influenced by the transmembrane domains of

FcγRIIA and FcγRIIB, which differ from each other in three amino acids and which were swapped along with the cytoplasmic domains in the chimeric receptors. The transmembrane domains of both receptors have been shown to affect their localization to membrane microdomains (i.e. lipid raft). The transmembrane domain of FcγRIIA is important in regulating its inducible association with lipid rafts and FcγRIIA-mediated responses like NFκB activation or phagocytosis (Garcia-Garcia et al., 2007). In contrast, endocytosis of soluble

immune complexes by FcγRIIA is less dependent on lipid rafts (Vieth et al., 2010). An SLE- associated polymorphism with a single transmembrane amino acid substitution in FcγRIIB1 results in its exclusion from lipid rafts and an impairment of its inhibitory function (Floto et al.,

2005).

Preferential degradation of the activating FcγRIIA coupled with persistence of its inhibitory counterpart implies that after immune complex stimulation, not only does the level of the activating receptor decline, but the ratio of activating to inhibitory receptor also decreases.

This reduction should serve to accentuate the termination of signalling from activating receptors, while maintaining the cell's ability to clear immune complexes. Thus, these fundamental differences in the sorting of FcγRIIA and FcγRIIB2 add an additional level of modulation of signalling over the longer term beyond the initial downregulation of activating signals by ITIM-mediated dephosphorylation.

3.5 Appendix

Co-localizations Analysis of Two or Three Molecules by Fluorescence Microscopy

In this thesis, in order to investigate the trafficking of FcγRII and ligands to various endocytic compartments, the co-localization of these molecules with different endosomal markers was examined by immunofluorescence and microscopy. As shown in Appendix Fig. 1, not only is the strength of staining signal in each channel and the amount of internalized agIgG heterogenous between cells, different parts of lysosomal compartments also show variations in

LAMP1 staining within a cell. Due to these factors, colocalized signals display a range of color from magenta to green in the merged images. Only when the strengths of the two staining signals are relatively the same do the co-localized puncta appear white. Therefore, besides examining the merged images, it is important to compare individual channels when studying

co-localization as merged color alone can be misleading. For example in Appendix Fig. F3, although the cell appears very magenta in the merged image, extentive co-localization of agIgG with Lamp1 can be seen when individual channels are compared. Thus, in this thesis, individual channels as well as merged images are shown for all the co-localization experiments.

It should be noted that in this particular experiment, although most internalized agIgG co- localizes with LAMP1 after 80 minutes of FcγRIIA engagement, not all LAMP1-positive compartments contain agIgG.

A LAMP1 agIgG LAMP1/agIgG

B1 B2 B3

C1 C2 C3

D1 D2 D3

E1 E2 E3

F1 F2 F3

Fig. 2-1 Microscopy analysis of co-localization of agIgG internalized by FcγRIIA to LAMP1. The cells shown in this figure are in the same field as those in Fig. 3-1 A-C. ts20 cells expressing MH-tagged FcγRIIA were incubated with agIgG for 80 min. agIgG was detected by immunofluorescence with anti-human IgG antibody (B2, C2, D2, E2, F2). LAMP1 was localized with anti-LAMP1 antibody (B1, C1, D1, E1, F1). Merged images show agIgG in green and LAMP1 staining in magenta (A, B3, C3, D3, E3, F3). The image of the entire field of cells captured is shown in (A) and five cells were zoomed in to show co-colocalization (B-F). Arrows highlight instances of co-localization of agIgG and LAMP1. Open arrowheads in D2,3 and E2,3 indicate rare agIgG containing compartments that do not obviously colocalize with a LAMP1 positive compartment. Scale bar: 10 μm. Images were analyzed by widefield microscopy.

Chapter 4 Comparison of Endocytosis Mediated by FcγRIIA and FcγRIIB2

* Fig. 4-1 has been published in Journal of Biological Chemistry. 2006. Mero P, Zhang CY,

Huang ZY, Kim MK, Schreiber AD, Grinstein S, Booth JW. 2006. Phosphorylation- independent ubiquitylation and endocytosis of FcγRIIA. J Biol Chem. 281 (44): 33242-33249

* Fig. 4-2, Fig. 4-4 to Fig. 4-9 have been submitted to Molecular Immuonology

* Fig. 4-3 is unpublished data

4.1 Abstract

An important function of FcγRs is the removal of IgG-containing immune complexes from the circulation. The activating receptor FcγRIIA and inhibitory receptor FcγRIIB2 are both expressed on human myeloid cells, and are both capable of mediating endocytosis of immune complexes. I studied endocytosis of these two receptors expressed by transfection in ts20 Chinese hamster fibroblasts. I find that while induction of FcγRIIA-mediated endocytosis requires the participation of the ubiquitin-conjugating system, the endocytosis of FcγRIIB2 does not. FcγRIIB2, unlike FcγRIIA, does not become ubiquitylated upon engagement with multivalent immune complexes. FcγRIIB2 internalizes immune complexes at a faster rate than

FcγRIIA, and accelerates the endocytosis of FcγRIIA upon receptor co-engagement, allowing for a more rapid FcγRIIA downregulation from cell surface. This may represent a novel mechanism by which the inhibitory receptor can reduce signalling by the activating FcγRs.

4.2 Introduction

In the immune system, the recognition of multivalent IgG-containing complexes is carried out by FcγRs, key players in both the afferent and efferent phase of an immune response

(Nimmerjahn et al., 2008). These receptors are essential in allowing a range of effector responses including ADCC, DC maturation and inflammatory mediator production.

FcγRIIA and FcγRIIB are the activating and inhibitory members of the human FcγRII subfamily, respectively. Both receptors bind to the Fc portion of IgG with low affinities. The ratio of expressions of FcγRIIA to FcγRIIB on different myeloid cells varies and can be regulated by cytokines (Boruchov et al., 2005; Liu et al., 2005; Pricop et al., 2001;

Tridandapani et al., 2002). Present only in humans and other primates, FcγRIIA is widely expressed on leukocytes (Takai, 2005; Tan Sardjono et al., 2003). The intracellular domain of

FcγRIIA contains an ITAM that is phosphorylated by SFK upon receptor clustering to trigger downstream signalling cascades (Daeron, 1997). This motif is similar but not identical to the

ITAM in Fc receptor γ-chain, with a unique sequence of 12 instead of 7 amino acids in between the two YxxL motifs. Whereas there is 92% sequence identity in the extracellular domains of

FcγRIIA and FcγRIIB, the FcγRIIB intracellular domain diverges from that of FcγRIIA and contains an ITIM. When FcγRIIB is co-engaged with activating receptors, phosphatases are recruited to the ITIM and dampen the ITAM-mediated inflammatory response (Wang et al.,

2007). Therefore, the overall balance between positive and negative signals from these two receptors is important to maintain an appropriate level of effector responses to immune complexes. Imbalances in activating and inhibitory signalling causing exacerbation of FcγR- mediated effector responses have been associated with the manifestation of autoimmune disorders (Tan Sardjono et al., 2003).

One important role FcγRs play during the course of immune responses is the removal of

IgG-opsonized foreign material from the circulation. FcγRIIA is capable of inducing both phagocytosis of large antibody-coated particles and endocytosis of small soluble immune complexes (Booth et al., 2002; Indik et al., 1991). In contrast to phagocytosis, which requires

ITAM-mediated receptor phosphorylation and actin rearrangement, FcγRIIA endocytosis of soluble immune complexes is phosphorylation-independent and proceeds via clathrin-coated pits. The inhibitory FcγRIIB occurs as two isoforms generated by alternative splicing, with

FcγRIIB1 present in B cells and FcγRIIB2 mainly expressed in myeloid cells (Hunter et al.,

1998; Hunziker et al., 1994). Neither of the two isoforms possesses phagocytosis-inducing capability. However, human FcγRIIB2 is able to induce endocytosis of soluble immune complexes (Van den Herik-Oudijk IE et al., 1994). For the homologous mouse FcγRIIB, a di- leucine motif lying within the ITIM has been shown to be important for endocytosis (Hunziker et al., 1994; Matter et al., 1994).

Ubiquitylation has been shown to be an important process to regulate the trafficking of many membrane proteins (Shin et al., 2006). It has been implicated in the endocytosis of

EGFR and GHR (Stang et al., 2004; Staub et al., 2006; Strous et al., 1996). It was found that ubiquitylation is also important for endocytosis of soluble immune complexes mediated by

FcγRIIA (Booth et al., 2002).

FcγRIIA and FcγRIIB2 expressed on human myeloid cells are both competent for endocytosis. However, the extent to which endocytosis via these two receptors proceeds by similar or distinct mechanisms is unclear. Understanding the trafficking behaviours of these receptors when engaged by immune complexes will provide insights into the control of inflammatory signalling by multiple FcγRs. In this work, I compared the requirements for ubiquitylation and the internalization kinetics of these two endocytic pathways. I also

investigated how receptor endocytosis is affected when FcγRIIA and FcγRIIB2 are co-engaged.

I find that FcγRIIB2, in contrast to FcγRIIA, can internalize immune complexes in a ubiquitylation-independent manner. This endocytosis is more rapid than that mediated by

FcγRIIA, and co-engagement with FcγRIIB2 may allow for the acceleration of FcγRIIA internalization and downregulation.

4.3 Results

4.3.1 Identification of a region within the ITAM of FcγRIIA crucial for its endocytosis

Internalization motifs in the cytoplasmic domain of cell surface receptors are responsible for driving endocytosis induced by ligand binding. I first sought to identify by receptor mutagenesis the amino acids in the intracellular domain of FcγRIIA important for its internalization of immune complexes. Wild type or mutant FcγRIIA were expressed in Chinese hamster fibroblast ts20 cells by transfection and engaged with multivalent agIgG for endocytosis. A truncation mutant (IIAΔC) with 25 amino acids deleted from its C-terminus shows agIgG internalization by FcγRIIA into central punctate endosomes similar to wildtype receptor (Fig. 4-1A-C). This suggests that C-terminal residues of FcγRIIA containing the second YxxΦ domain of the ITAM are not required for FcγRIIA endocytosis.

Next, I examined the role of the region upstream of the FcγRIIA truncation, which contains the first YxxΦ domain of the ITAM (Fig. 4-1A). Single amino acids between E281 and N291 were replaced by alanine residues either in the context of the full-length receptor or of the IIAΔC that still had fully functional endocytosis, with the exception of Y287, which was mutated to phenylalanine. Three residues (Y287, M288 and L290) that lie within the FcγRIIA

ITAM are crucial to the induction of FcγRIIA-mediated endocytosis. Mutation of Y287 to phenylalanine abolished FcγRIIA-mediated agIgG internalization (Fig. 4-1I). These finding 99

A FcγRIIA intracellular domain ITAM

point mutations truncation (ΔC) 240 279 316 IIA IIAΔC IIA E281AΔC

B C D IIA T282AΔC IIA D284AΔC IIA G285AΔC

E F G IIA G286AΔC IIA Y287F IIA M288AΔC

H I J IIA T289A IIA L290AΔC IIA N291AΔC

K L M total agIgG/surface agIgG

Fig. 4-1 Identification of key residues in the FcγRIIA intracellular domain crucial for the induction of its endocytosis. (A) the protein sequence of FcγRIIA intracellular domain. Brown arrow points to the five lysine residues that can be potential ubiquitylation sites. The 11 residues in the box are those that were mutated one at a time to alanine or phenylalanine. The three residues highlighted in red are those crucial for the induction of FcγRIIA endocytosis. The arrow points to the position of FcγRIIA truncation. Upper bracket indicates the position of FcγRIIA ITAM motif. (B-M) Endocytosis of agIgG was analyzed in ts20 cells transfected with MH-tagged mutants of FcγRIIA and incubated with agIgG for 30 minutes. agIgG remaining outside (green) and total agIgG in the cell (red) were detected by fluorescence microscopy with fluorophore-conjugated anti-human antibody and yellow indicates co- localizations of the two. The merged images are shown. Images were analyzed by widefield microscopy. Representative of three experiments. 100

suggests that although SFK action is not necessary for the internalization (Mero et al., 2006) or degradation (Fig. 3-17) of FcγRIIA, the presence of Y287 is nonetheless required for its endocytosis. YxxΦ motifs can interact with AP2 and thereby act as targeting signals for endocytosis (Bonifacino et al., 2003). Consistent with the hypothesis that Y287MTL functions as an AP2 binding motif, mutation of L290 to alanine also impaired endocytosis whereas mutation of T289 to alanine had no effect (Fig. 4-1K, L). However, M288 is also required for endocytosis as mutation of this residue to alanine inhibited endocytosis (Fig. 4-1J). Other residues in the vicinity of Y287MTL including E281, T282, G285,G286 and N291 are not important for FcγRIIA endocytosis as judged from mutagenesis to alanines whereas mutation of

D284 seems to have a partial inhibitory effect (Fig. 4-1D-H, M).

4.3.2 Endocytosis of FcγRIIB2 does not require ubiquitylation

It has been shown previously using ts20 cells transfected with FcγRIIA that the ubiquitin conjugation system is required for the induction of FcγRIIA-mediated endocytosis

(Booth et al., 2002). I sought to test whether FcγRIIB2-mediated endocytosis similarly requires the participation of the ubiquitylation system. ts20 cells carry a temperature-sensitive mutation in the E1 ubiquitin-activating enzyme that allows for inactivation of all cellular ubiquitylation by incubating cells at a restrictive temperature. Accordingly, I pre-incubated ts20 cells transfected with either FcγRIIA or FcγRIIB2 at a non-permissive temperature of 42.5oC to inactivate the E1 enzyme. AgIgG was then added to the cells to trigger endocytosis. AgIgG internalized by either FcγRIIA or FcγRIIB2 was delivered to central punctate endosomes (Fig.

4-2A, D). Whereas agIgG uptake was blocked upon abolition of ubiquitylation in FcγRIIA- expressing cells, FcγRIIB2-mediated agIgG endocytosis was unaffected (Fig. 4-2B, E).

Treatment of FcγRIIA-expressing cells with the proteasome inhibitor MG132 also blocks

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Fig. 4-2 Endocytosis of FcγRIIB2 does not require ubiquitylation. Endocytosis of agIgG was analyzed in ts20 cells transfected with MH-tagged FcγRIIA (A-C) or FcγRIIB2 (D-F). AgIgG was added for 30 minutes with (B, E) or without (A, C) pre-incubation of the cells for 2 hours at a non-permissive temperature (42.5oC) to inactivate the E1 ubiquitin activating enzyme. Alternatively, cells were pretreated with the proteasome inhibitor MG132 for 2hrs to deplete free ubiquitin (C, F). AgIgG remaining on the cell surface (green) and total agIgG (red) were detected with fluorophore-conjugated anti-human antibodies and yellow indicates co- localizations of the two. The merged images are shown. Scale bar: 10 μm. Images were analyzed by widefield microscopy. Representative of five experiments. control +MG132 RIIA γ Fc

AB RIIB2 γ Fc CD

Fig. 4-3 MH tag does not affect the initial induction of FcγRIIA- or FcγRIIB2-mediated endocytosis. Untagged FcγRIIA (A, B) or FcγRIIB2(C, D) expressing ts20 cells were incubated with agIgG for 30 minutes at 34oC after pre-treatment with (B, D) or without (A, C) MG132 for 2 hours. AgIgG remaining outside (green) and total agIgG (red) were detected and yellow indicates co-localizations of the two. The merged images are shown. Scale bar: 20μm. Images are representative of three experiments. 102

FcγRIIA endocytosis (Fig. 4-2C). This is likely by virture of an indirect effect through depletion of the cellular pool of free ubiquitin, since proteasome inhibitor treatment blocks receptor ubiquitylation (Mero et al., 2006). In contrast, the internalization of agIgG by

FcγRIIB2 was not prevented by treatment with MG132 (Fig. 1F). This divergent effect of

MG132 on FcγRIIA- and FcγRIIB2-mediated endocytosis was also observed in ts20 cells expressing untagged receptors, implying that the difference is not due to effects of the MH tag

(Fig. 4-3).

4.3.3 FcγRIIA, but not FcγRIIB2, is ubiquitylated upon receptor engagement

Engagement of FcγRIIA with agIgG results in ubiquitylation of the receptor in a manner independent of receptor phosphorylation (Booth et al., 2002; Mero et al., 2006). Here, I tested whether FcγRIIB2 is similarly ubiquitylated after receptor ligation by performing immunoprecipitation of receptors expressed in ts20 cells followed by western blotting. As shown in Fig. 4-4, while FcγRIIA became ubiquitylated upon addition of agIgG, little if any

FcγRIIB2 ubiquitylation was detected.

4.3.4 Rate of endocytosis mediated by FcγRIIA and FcγRIIB2

In light of these differences in both receptor ubiquitylation and the requirement for ubiquitylation for internalization, I tested whether the two endocytic pathways also differ in the kinetics of receptor internalization. ts20 cells that express FcγRIIA or FcγRIIB2 at similar levels (the same cells as in Fig. 3-2B) were incubated with agIgG for different lengths of time followed by detection of internalized agIgG by microscopy. Whereas little internalized agIgG was observed after 10 minutes of incubation in FcγRIIA-expressing cells, agIgG was delivered by FcγRIIB2 to endosomal compartments within 5 minutes (Fig. 4-5A-H). Quantitation of

103

Fig. 4-4 FcγRIIA but not FcγRIIB2 becomes ubiquitylated during the induction of endocytosis. ts20 cells expressing MH-tagged FcγRIIA or FcγRIIB2 were incubated with (lane 2, 4) or without (lane 1, 3) agIgG for 10 minutes. Following lysis of the cells, the receptors were immunoprecipitated and blotted with anti-ubiquitin antibodies (top blot). The blot was reprobed with anti-myc antibodies to detect receptors (bottom blot). Positions of 130 and 55 kDa markers are indicated. Bracket indicates positions of ubiquitylated receptors. Triangle indicates positions of unmodified FcγRIIA. Arrow indicates positions of unmodified FcγRIIB2. Representative of three experiments.

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0 min 5 min10 min 30 min

IIA

A BCD

IIB

EFGH

I internalized agIgG 35 30 IIA G IIB2 Ig 25

l a 20

ta 15

f to

o 10 % 5 0

Fig. 4-5 FcγRIIB2 endocytosis of agIgG is more rapid than that of FcγRIIA. (A-F) ts20 cells expressing MH-tagged FcγRIIA (A-D) or FcγRIIB2 (E-H) were stimulated with agIgG for 0 minutes (A, E), 5 minutes (B, F), 10 minutes (C, G) or 30 minutes (D,H). After fixation and cell permeabilization, agIgG was detected with Cy3-conjugated secondary antibody and analyzed by widefield microscopy. Scale bar: 10 μm. Images are representative of three experiments. (G) ts20 cells expressing MycHis-tagged FcγRIIA (blank bar) or FcγRIIB2 (grey bar) were incubated with FITC-agIgG on ice, then washed and incubated at 34oC for 20 minutes. Internalized agIgG was quantified by flow cytometry after quenching of surface FITC-agIgG with trypan blue. The amount of the internalized agIgG is expressed as a percentage of the mean fluorescence intensity of total surface bound FITC-agIgG without trypan blue quenching after background subtraction of fluorescence intensity observed with quenching at time 0. More FITC-agIgG binding to FcγRIIB2-expressing cells was observed at time 0 compared to cells expressing FcγRIIA.

105

agIgG uptake by flow cytometry also showed a substantially greater fraction of surface-bound agIgG was taken up by FcγRIIB2 within 20 minues (Fig. 4-5I).

I also analyzed the kinetics of internalization by cross-linking receptors with anti-FcγRII antibody AT10 (which binds either receptor) and following the internalization of cross-linking antibody by flow cytometry. Consistent with the observations of agIgG uptake by microscopy,

FcγRIIB2 exhibits a faster initial rate of internalization than FcγRIIA when engaged with AT10, both in the presence or absence of secondary antibody cross-linking (Fig. 4-6). More than 40% of AT10 bound to FcγRIIB2 was internalized within the first 10 minutes of receptor engagement, after which net disappearance from the surface reached a plateau. This stands in contrast to FcγRIIA, where less than 20% of its uptake was observed during this time period.

Importantly, we confirmed that decreases in the levels of surface AT10 antibody complexes over time are due to their internalization rather than release from the cell, as the total amount of cell-associated complexes remains constant (Fig. 4-7). FcγRIIB2 internalization was similar with or without receptor clustering with secondary antibody, whereas the rate of FcγRIIA internalization was increased by secondary antibody. The plateau at later times for FcγRIIB2 presumably reflects the fact that FcγRIIA is directed to lysosomes under these conditions while

FcγRIIB2 recycles (Chapter 3). The faster initial drop in FcγRIIB2 and the fact that addition of clustering secondary antibody does not change the rate of internalization suggests that immune complex uptake via FcγRIIB2 occurs by their binding to receptors that are constitutively cycling. Moreover, the rate of internalization through this pathway is faster than that of clustered FcγRIIA.

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A surface-bound antibody B surface-bound antibody complex 120 120 100 100 IIA 1:4

I I IIB2 1:4

F 80 F

M M 80

i 60 i

f 40 f 40

IIA 1:0 % 20 % IIB2 1:0 20 0 0 0 10203040 0 10203040 time (min) time (min)

Fig. 4-6 FcγRIIB2 endocytosis of antibody complexes is more rapid than that of FcγRIIA. ts20 cells expressing MH-tagged FcγRIIA or FcγRIIB2 were incubated with AT10 at 4oC with (B) or without (A) subsequent incubation at 4°C with Alexa488 goat anti-mouse secondary antibodies to cross-link receptors. Cells were fixed after being chased at 34oC for the indicated times. Receptor internalization was measured by the disappearance of prebound AT10 antibodies from the cell surface by flow cytometry. Uncrosslinked AT10 remaining on the cell surface was detected by immunofluorescence with Cy5-anti-mouse antibodies. Cross-linked AT10 antibody complexes were detected with Cy5-anti-goat antibodies. Receptor internalization is expressed as a percentage of the initial mean fluorescence intensity of surface- bound Cy5-labeled antibodies at time 0 after background subtraction of fluorescence intensity of untransfected ts20 cells. Triangles or squares with dashed line: FcγRIIA; Diamonds or crosses with solid line: FcγRIIB2. Error bars indicate s.d. n = 3.

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ts20-FcγRIIA

A B

ts20-FcγRIIB2

C D

monocytes plus IL-4/IL-10

E F

% % total AT10 antibody complexes surface antibody complexes

Fig. 4-7 Surface ligands are not released to the medium after FcγRII engagement in warm medium. ts20 cells expressing MycHis-tagged FcγRIIA (A, B) or FcγRIIB2 (C, D) or human monocytes were treated overnight with IL-4 and IL-10 (E, F) were incubated with AT10 at 4oC with subsequent incubation at 4°C with Alexa488 labeled goat anti-mouse secondary antibodies (A, C, E) to cross-link receptors. Cells were then chased at 34oC for 30 minutes (dotted line) or without chasing (solid line) before fixation. The cells were chased at 34oC for the indicated times, and AT10 antibody complexes remaining at the cell surface were detected by flow cytometry using Cy5- anti-goat antibodies (B, D, F). Shaded histogram: untransfected ts20 cells (A-D); IL-4 and IL-10 treated human monocytes incubated with secondary antibodies in the absence of AT10 antibody n = 3.

108

4.3.5 Acceleration of FcγRIIA internalization by FcγRIIB2

If FcγRIIB2 is internalized more rapidly than FcγRIIA, this suggests the possibility that

FcγRIIB2 could accelerate the internalization of FcγRIIA if the two receptors are co-engaged by immune complexes, as will occur in cells that express both receptors. To test this idea, I transiently transfected untagged FcγRIIB2 into ts20 cells stably expressing MH-tagged

FcγRIIA. After incubation with agIgG, the subcellular localizations of FcγRIIA, FcγRIIB2 and agIgG were analyzed by immunofluorescence. FcγRIIA internalization was indeed accelerated in the presence of FcγRIIB2 (Fig. 4-8A-E). FcγRIIA was now observed in endosomes, colocalized with FcγRIIB2 and agIgG within 10 minutes of agIgG addition. To provide further support for the idea that FcγRIIB2 can accelerate FcγRIIA internalization, I next tested whether

FcγRIIB2 is able to induce the internalization of FcγRIIA under conditions where FcγRIIA endocytosis is normally inhibited. FcγRIIA-mediated endocytosis is dependent on tyrosine residue 287 within the FcγRIIA intracellular domain (Fig. 4-1A). When FcγRIIB2 was transiently transfected into cells expressing a mutated FcγRIIA in which this residue was changed to phenylalanine, the mutated FcγRIIA was delivered to endosomal compartments only in the FcγRIIB2-expressing cells (Fig. 4-8F-J). Similarly, while pre-treatment of cells with MG132 completely blocked FcγRIIA-mediated endocytosis (Fig. 4-2C), in cells transiently expressing FcγRIIB2, the FcγRIIB2 was capable of delivering FcγRIIA along with agIgG into endosomes despite the presence of the inhibitor (Fig. 4-8K-P).

4.3.6 FcγRIIB2 upregulation results in an increase in the rate of FcγRII internalization in monocytes

The relative levels of expression of FcγRIIA and FcγRIIB in human myeloid cells are modulated by the cytokine milieu (Pricop et al., 2001). In particular, the combination of IL-4

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agIgG IIA-MH IIB2 IIA-MH/IIB2 IIA-MH/agIgG G -M 10 min

A BCDE agIgG IIA-Y2F-MH IIB2 IIA-Y2F-MH/IIB2 IIA-Y2F-MH/agIgG -MG 40 min

F GH I J agIgG IIA-MH IIB2 IIA-MH/IIB2 IIA-MH/agIgG +MG 30 min K LMNP

Fig. 4-8 Acceleration of FcγRIIA internalization by FcγRIIB2. ts20 cells stably expressing MH-tagged wild type FcγRIIA (A-E, K-P) or endocytosis-incompetent FcγRIIA mutant with tyrosine 287 mutated to phenylalanine (Y287F) (F-J) were transfected with untagged FcγRIIB2. After pretreating cells with (K-P) or without (A-J) proteasome inhibitor MG132 for 2 hours, the cells were incubated with agIgG for 10 minutes (A-E), 30 minutes (K-P) or 40 minutes (F-J) for endocytosis. AgIgG, FcγRIIA, and FcγRIIB2 were detected by immunofluorescence using anti-human (A, F, K), anti-myc (B, G, L), and rabbit anti-FcγRIIB2 (C, H, M) antibodies, respectively. (D, I, N) Merged images show FcγRIIA in red and FcγRIIB2 in green. (E, J, P) Merged images show FcγRIIA in red and agIgG in green. Yellow in merged images indicates co-localizations of wildtype or mutated FcγRIIA-MH with agIgG. Arrows highlight instances of co-localization. Hollow arrow heads indicate cells expressing only FcγRIIA-MH. Scale bar: 10 μm. Images were analyzed by widefield microscopy. Representative of three experiments.

110

and IL-10 results in upregulation of FcγRIIB levels in monocytes without any change in the level of FcγRIIA (Su et al., 2007; Wijngaarden et al., 2004). Using the pan-FcγRII antibody

AT10 (which recognizes both FcγRIIA and FcγRIIB), I examined the kinetics of internalization of FcγRII in primary human monocytes with or without upregulation of FcγRIIB by this cytokine treatment. Consistent with the findings by Wijingaarden et al., with combined treatment of IL-4 and IL-10, the suface expression of FcγRII (i.e. FcγRIIA and FcγRIIB) doubled (Fig. 4-9A, B), presumably due to the upregulation of FcγRIIB. I found that the rate of internalization of total FcγRII was higher after cytokine-mediated FcγRIIB upregulation than in untreated cells (Fig. 4-9B). This is consistent with my findings in the ts20 transfectant model that FcγRIIB is internalized more rapidly than FcγRIIA.

4 Discussion

I demonstrate in this study that the endocytic mechanisms used by the two members of the human FcγRII family are fundamentally different. FcγRIIA-mediated endocytosis requires the participation of the ubiquitin conjugation system whereas FcγRIIB2-mediated endocytosis does not. In both transfected cells and primary human monocytes, I find that FcγRIIB2 is internalized at a faster rate than FcγRIIA. Moreover, FcγRIIB2 can have a dominant effect when co-engaged with FcγRIIA, accelerating its uptake.

A region in ITAM of FcγRIIA including Y287, M288 and L290 is crucial for the induction of FcγRIIA-mediated endocytosis. These residues have also been shown to be important for the ubiquitylation of FcγRIIA (Mero et al., 2006). The Y287MTL region is not likely to be an YXXΦ motif that targets transmembrane proteins for endocytosis and lysosomal sorting because of the requirement of M288 for FcγRIIA endocytosis. Furthermore, while

YXXΦ motifs mediate endocytosis by interacting with clathrin-associated adaptor protein AP-2,

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A B 100 3 - IL4/IL10 2.5

80 +IL4/IL10

o e

t 2

d im

60 e t

t 1.5

liz

n a

40 e m

m 1

e n

x 20

r 0.5

m n M 0 f 0 2 o 3 4

100 101 10 10 10 0 10203040 % time (min) AT10

Fig. 4-9 FcγRIIB upregulation in human monocytes results in an increase in the rate of internalization of cross-linked FcγRII. (A) Human monocytes were treated with (solid line) or without (dashed line) IL-4 or IL-10 overnight to allow for FcγRIIB upregulation. Total FcγRII on the cell surface was detected with anti-FcγRII antibody AT10 and secondary Alexa488-labelled goat anti-mouse secondary antibody (solid line and dashed line). Cells without AT10 staining was shown (shaded histogram). (B-D) Monocytes were incubated with AT10 at 4oC followed by cross-linking at 4°C with Alexa488 goat anti-mouse secondary antibodies. After the cells were chased at 37oC for the indicated times, AT10 antibody complexes remaining at the cell surface were detected by flow cytometry with Cy5- anti-goat antibodies. Receptor internalization is expressed as the ratio of the mean fluorescence intensity of surface-bound AT10 antibody complexes to that of monocytes not treated with cytokines at time 0. Open squares: with IL-4 and IL-10 treatment; Solid diamonds: no IL-4 and IL-10 treatment. Error bars indicate s.d. n = 3.

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FcγRIIA endocytosis is AP2 independent (Mero et al., 2006). Beside these three residues in the

ITAM, D284 also plays a role, albeit not an essential one, in the initiation of FcγRIIA endocytosis. These findings point to the existence of a novel endocytic signal for this transmembrane receptor.

When the ubiquitin-conjugating system is shut down through incubation of cells at non- permissive temperature, I observed that endocytosis of FcγRIIA, but not FcγRIIB2 is blocked.

The Booth lab has shown previously that ubiquitylation of FcγRIIA occurs prior to the assembly of clathrin-coated pits in a manner independent of receptor phosphorylation by SFK

(Mero et al., 2006). As phosphorylation of receptors is generally considered to be required for triggering receptor ubiquitylation, these findings point to a novel mechanism of FcγR endocytosis.

FcγRIIA-mediated endocytosis requires the participation of ubiquitin-conjugating system and the receptor itself is ubiquitylated upon clustering with multivalent ligands (Fig. 4-2, 4-4).

However, none of the five lysine residues in the intracellular domain of FcγRIIA are absolutely required for either the internalization or the lysosomal degradation of the receptor (Fig. 3-21).

One possibility is that FcγRIIA ubiquitylation occurs at the non-lysine residues in its intracellular domain. It has been shown that MHC class I lacking lysine residues can be ubiquitylated at a cysteine residue on its cytoplasmic tail leading to its degradation by a viral E3 ubiquitin ligase (Cadwell et al., 2005). Another group demonstrated the ubiquitylation of MHC class I heavy chain at the serine, threonine, or lysine residues on its cytoplasmic tail, each of which is sufficient to induce endoplasmic reticulum-associated degradation of the heavy chain

(Wang et al., 2007). FcγRIIA has only one cysteine residue in the N-terminus of its intracellular domain (Fig. 1-3B). Due to its close proximity to the transmembrane region of the receptor, it is unlikely that this cysteine residue serves as a ubiquitylation site. There are three

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serines and five threonine residues in the cytoplasmic tail of FcγRIIA. Further investigation is needed to test if these residues can be potential ubiquitylation sites.

There are two possibilities that may account for the inhibition of FcγRIIA-mediated endocytosis of agIgG by pretreatment with proteasome inhibitor MG132: 1) via direct abolishment of proteasome activities; 2) via blocking receptor ubiquitylation indirectly. I found that at least an hour of pre-incubation is required before the addition of agIgG for MG132 to have any negative effect on FcγRIIA endocytosis. Thus, the possibility of MG132 acting via acute inhibition of proteasome activity is ruled out. Furthermore, MG132 also abolishes clustering-induced FcγRIIA ubiquitylation by western blotting with anti-ubiquitin antibody, suggesting a direct association between FcγRIIA-mediated endocytosis and receptor ubiquitylation (Mero et al., 2006). Thus, inhibition of proteasome activation by MG132 treatment indirectly affects FcγRIIA receptor ubiquitylation required for the induction of

FcγRIIA endocytosis possibly by depletion of free ubiquitin in the cytosol.

I observed that FcγRIIA was internalized at a slower rate than FcγRIIB2 upon immune complex binding (Fig. 4-5 to Fig. 4-6). This difference in the rate of receptor internalization may result from the divergent requirement of the two endocytic pathways for ubiquitylation. A dileucine motif in the cytoplasmic domain of murine FcγRIIB has been shown to be important for its endocytosis (Hunziker et al., 1994; Matter et al., 1994). My findings indicate that the cellular ubiquitylation machinery is not required for endocytosis of human FcγRIIB2, and moreover that internalization occurs in a manner that does not require significant receptor clustering (beyond potential dimerization induced by bivalent anti-FcγRII antibodies). These results suggest that the dileucine motif in human FcγRIIB2 is sufficient to drive constitutive internalization of the receptor and any associated bound immune complexes. In contrast, for

FcγRIIA, receptor clustering both triggers ubiquitylation and increases the rate of receptor

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internalization. Ubiquitylation secondary to receptor clustering may thus be a rate-limiting step for internalization in the case of FcγRIIA. It has been noted that in studies in transfected B cells, van den Herik-Oudjik et al found in contrast a more rapid internalization of human IgG aggregates by FcγRIIA than FcγRIIB2 (Van den Herik-Oudijk IE et al., 1994). Whether this relates to differences in methodology or to the cell type of expression is unclear.

Upon cross-linking with antibody complexes, FcγRIIB2 loss from the cell surface occurs in the first 10 minutes, after which surface levels become stable (Fig. 4-6B). In contrast, a continuous decrease in the surface level of FcγRIIA was observed throughout 30 minutes (Fig.

4-6B). These differences are consistent with my findings in Chapter 3 that FcγRIIA and

FcγRIIB2 are sorted through different routes after their internalization. FcγRIIB2 are sorted into recycling endosomes. The plateau in level of surface antibody-bound-FcγRIIB2 suggests that a dynamic equilibrium has been reached between the surface pool of FcγRIIB2 and those in recycling endosomes. On the other hand, FcγRIIA takes a longer time to be internalized into endosomal compartments, and is removed from the cell surface and degraded in late endosomal/lysosomal compartments.

In this thesis, I also examined the internalization rate of FcγRIIA and FcγRIIB in primary human monocytes by altering the relative expression of the two receptors with cytokine treatment. Studies have shown that FcγRIIB is expressed on human monocytes at a much lower level than FcγRIIA (Boruchov et al., 2005; Su et al., 2007). This finding implies that it is mainly FcγRIIA that is engaged on these cells by incubation with anti-FcγRII antibody AT10

(Fig. 4-9B). The level of FcγRIIB increases in the presence of IL-4 plus IL-10 while FcγRIIA expression is not affected (Wijngaarden et al., 2004). Thus, as the overall FcγRII expression doubled after such cytokine treatment (Fig. 4-9B), the ratio of expression of FcγRIIA to

FcγRIIB is expected to reach approximately 1:1. The faster rate of internalization of total

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FcγRII after FcγRIIB upregulation is consistent with my findings in ts20 cells that FcγRIIB takes up immune complexes more rapidly than FcγRIIA. However, further studies are needed to show whether the presence of FcγRIIB can increase the rate of FcγRIIA removal from the cell surface in response to immune complexes in human monocytes.

The acceleration of FcγRIIA internalization upon co-engagement with FcγRIIB2 has the potential to reduce the duration and magnitude of the activating signal mediated by FcγRIIA, in light of the fact that internalization of FcγRIIA leads ultimately to its degradation (Chapter 3).

This suggests that the inhibitory receptor FcγRIIB2 can downregulate cell signalling through two fundamentally distinct mechanisms. On one hand, ITIM-mediated recruitment of phosphatases directly antagonizes ITAM signalling. A second effect, however, may be that the presence of inhibitory receptors facilitates removal of the activating receptors from the cell surface. Such changes in the kinetics of receptor downregulation may be an important factor influencing signalling outcomes in many situations in which activating and inhibitory receptors are engaged at the cell surface.

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Chapter 5 General Discussion and Conclusion

5.1 Thesis overview

The aim of this thesis is to examine the trafficking behavior of FcγRIIA and FcγRIIB2 upon engagement with immune complexes. In chapter 3, I investigated the intracellular sorting of receptors and ligands after internalization via endocytosis. Chapter 4 of this thesis describes the requirements for ubiquitylation and internalization kinetics of FcγRIIA- or FcγRIIB2- mediated endocytosis. The results reveal fundamental differences in the trafficking of FcγRIIA and FcγRIIB2 during the initial induction of endocytosis and the subsequent intracellular sorting.

As illustrated in Fig. 5-1, upon clustering with immune complexes on the plasma membrane, FcγRIIA becomes ubiquitylated on its cytoplasmic tail. FcγRIIA ubiquitylation triggers its internalization into early endosomal compartment. As endosomal maturation progresses, FcγRIIA and immune complexes are both delivered into late endosomes/lysosomes for degradation. After the engagement of FcγRIIB2 for endocytosis, ligand-bound FcγRIIB2 is internalized at a rate faster than that of FcγRIIA into early endosomes without the requirement for ubiquitylation. The sorting route of the receptor diverges from that of the immune complexes at a certain stage of endosomal maturation. Immune complexes are sorted for lysosomal degradation whereas FcγRIIB2 recycles back to the cell surface through recycling endosomes. In cells where FcγRIIA and FcγRIIB2 are both expressed, these two receptors can be simultaneously co-engaged by multivalent immune complexes before their delivery into early endosomes. Furthermore, FcγRIIB2 is able to accelerate the rate of FcγRIIA endocytosis.

Immune complexes dissociate from both receptors before the sorting event occurs. This allows

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Fig. 5-1 Schematic representation of FcγRIIA and FcγRIIB2 trafficking when engaged or co-engaged with immune complexes A) As FcγRIIA is cross-linked by agIgG on the cell surface, FcγRIIA is ubiquitylated followed by internalization of receptors and immune complexes into early endosomes. As endosomal maturation proceeds, the ubiquitylated FcγRIIA and immune complexes are targeted into the internal vesicles of MVBs and get degradated. B) Following FcγRIIB2-mediated endocytosis of immune complexes, ligands are released from receptors. Immune complexes are sorted into MVB and then lysosomes to be degraded while FcγRIIB2 is recycled back to the cell surface via recycling endosomes. C) Upon co-engagement of FcγRIIA and FcγRIIB2, immune complexes are released before FcγRIIA and FcγRIIB2 are sorted independently.

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for independent routing of FcγRIIA and FcγRIIB2 to their distinct fates—FcγRIIA is still degraded in lysosomes where FcγRIIB2 accesses a non-degradative pathway.

The suppression of activating signals by FcγRIIA is known to be mediated by the inhibitory FcγRIIB2 through ITIM-mediated recruitment of phosphatases (Fig. 5-2). The differential trafficking of FcγRIIA and FcγRIIB2 provides an additional mechanism to downregulate FcγRIIA-mediated activating signals. The continuous redirection of FcγRIIA trafficking to lysosomes for degradation during ligand-induced endocytosis results in decreases in surface and overall levels of FcγRIIA. In contrast, the surface level of FcγRIIB2 does not change as the internalized FcγRIIB2 recycles back to the cell surface. Thus, the surface ratio of the activating FcγRIIA to the inhibitory FcγRIIB2 declines over the course of immune responses. This leads to the dampening of pro-inflammatory responses while the clearance of immune complexes in the circulation is ensured. FcγRIIB2 is more efficient in taking up immune complexes than FcγRIIA. The ability of FcγRIIB2 to accelerate the rate of FcγRIIA internalization upon co-engagement may serve as another way to reduce the duration and magnitude of activating signals by FcγRIIA. As FcγRIIA becomes more rapidly removed from cell surface, its lysosomal degradation is also quickened and its signalling duration shortened.

This dual system of signalling modulation provides interesting insights in the association between receptor trafficking and receptor signalling. Through regulating the internalization and sorting of different FcγRs, cells may shift the overall balance of activating and inhibitory receptors, and thus, change the overall signalling outcome.

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Fig. 5-2 Two mechanisms of downregulation of FcγRIIA-mediated activating signals by FcγRIIB2 When FcγRIIB2 is co-engaged with activating receptors such as FcγRIIA, activating signals can be down-regulated by FcγRIIB2 through two mechanisms: 1) ITIM-mediated recruitment of phosphatases by FcγRIIB2; 2) Differential sorting of FcγRIIA and FcγRIIB2 in response to immune complex engagement. The continuous degradation of FcγRIIA in lysosomes and recycling of FcγRIIB2 to the cell surface leads to a decrease in the surface ratio of the activating FcγRIIA to the inhibitory FcγRIIB2 over time.

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5.2 Characterization of FcγRIIA and FcγRIIB2 trafficking in primary human myeloid lineage cells

5.2.1 The complexity of human myeloid cells--various DC and macrophage subsets

In this thesis, the trafficking of FcγRIIA and FcγRIIB2 upon endocytosis was first characterized in Chinese hamster fibroblast ts20 cells heterologously expressing FcγRIIA or

FcγRIIB2 by transfection. Such a transfected cell model has the advantage of allowing for the examination of one FcγR-mediated endocytic pathway in isolation. In addition, it provides a means for me to elucidate the underlying molecular mechanism by transfection of mutant versions of receptors into these cells.

Compared to the transfected cell model, primary human ex vivo differentiated myeloid cells have the advantage of endogenously expressing FcγRII, providing natural cellular environments for FcγR activity. In this thesis, I have explored the mechanisms of internalization and intracellular sorting of antibody complexes bound to FcγRIIA in primary human myeloid cells including monocytes, macrophages and moDCs. However, it remains unclear how FcγRs behave in human cells and whether their properties differ from one cell type to another. Increasing evidence has shown that both human and mouse iDCs with more alkaline phagosomal and endosomal compartments and less efficient lysosomal proteolysis have limited capacity for antigen degradation after internalization compared to mDCs, macrophages and even fibroblasts (Delamarre et al., 2005; Trombetta et al., 2003).

Furthermore, it has been shown that the increase in the alkalinity of the phagolysosome leading to limited proteolysis iDCs is regulated by NOX2 (Jancic et al., 2007; McCurley and Mellman,

2010; Savina et al., 2006). However, the implication of altered endosomal function for membrane receptor trafficking in iDCs has not been addressed. It is conceivable that these receptors persist longer along with their ligands and are processed differently by iDCs, resulting

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in different fates for these receptors, e.g. trafficking of intact receptors and ligands to the cell surface in iDCs instead of immediate degradation in macrophages. Furthermore, monocytes can be differentiated into different types of macrophages by various cytokine treatments. GM-

CSF induced “type I” macrophages are different in several respects from M-CSF induced “type

II” macrophages (Fleetwood et al., 2007). They produce different cytokines and also display more rapid IκBα degradation and faster NFκB DNA binding in response to LPS, and thus are more pro-inflammatory.

5.2.2 Challenges of studying FcγRII trafficking in primary human myeloid cells

As ts20 cells do not express FcγRs endogenously, it is important to understand how immune complexes and FcγRII are transported in primary human myeloid cells. In order to approach this, there are several challenges to overcome.

The first challenge lies in choosing a type of human myeloid cells ideal for studying

FcγRII trafficking (i.e. the ones preferably expressing both FcγRIIA and FcγRIIB at detectable levels). In this thesis, I have investigated the trafficking of antibody complexes internalized by

FcγRIIA in three types of primary human ex vivo-differentiated myeloid cells: monocytes, monocyte-derived macrophages and moDCs. Monocytes were treated with IL-4 and IL-10 overnight to upregulate the expression level of FcγRIIB as freshly isolated monocytes express much less FcγRIIB than FcγRIIA (Joshi et al., 2006). moDCs have been shown to express no

FcγRs other than FcγRIIA and FcγRIIB (Boruchov et al., 2005). Consistent with previous reports (Guriec et al., 2006; Liu et al., 2006; Su et al., 2007), I found that the surface expression of FcγRIIA by IV.3 staining is comparable among different human myeloid cells (Table 3-1).

There are sufficient amounts of FcγRIIA in these cells for clear visualization of antibody- complex-containing endosomes. However, the problem lies in finding a human myeloid cell

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model that expresses FcγRIIB homogeneously and at a detectable level for trafficking studies.

The expression level of FcγRIIB in monocyte-derived macrophages is so low that it can hardly be detected by microscopy. The FcγRIIB expression in both monocytes and moDC varies at the cell-to-cell level and also among donors. Moreover, a very recent study suggests that moDCs exhibit higher capacity for lysosomal proteolysis compared to directly-isolated human

DCs; this raises the question of whether moDCs are in fact the best model for studying human

DC cell biology as differences in the proteolytic capability and alkalinity of endo-lysosomal compartments may impact sorting routes of receptor trafficking (McCurley et al., 2010).

Although these heterogeneities add an additional layer of complexity to the study of FcγRIIB trafficking, they may also reveal some biological features of FcγRIIB that are worth further investigation (e.g. the sensitivity of FcγRIIB expression towards various activation or differentiation signals). As FcγRIIB is the only inhibitory FcγR to downregulate immune complexes-mediated inflammatory responses, well-controlled FcγRIIB expression has both biological and clinical implications in the modulation of myeloid cell functions and the prevention of autoimmunity. For the study of FcγRIIB trafficking behaviour in human myeloid cells, one approach to generate a homogenenous expression of FcγRIIB at a detectable level in these cells is through retroviral transduction.

Another difficulty we face for characterizing FcγRIIB expression in human myeloid cells or their endocytosis after specific engagement is the lack of commercially available antibody specific for its extracellular domain. Multiple classes of FcγRs expressed on human myeloid cells may be co-engaged by immune complexes, making it difficult to tease out the mechanism of each FcγR-mediated endocytic pathway. In such case, antibodies that specifically bind to one class of FcγRII can be used. IV.3, an antibody against FcγRIIA extracellular domain, is commonly used as a FcγRIIA blocker. However, because of the high identity in the

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extracellular domains of FcγRIIA and FcγRIIB, it is not until recently that anti-FcγRIIB antibodies were generated. These antibodies include 2B6 (Boruchov et al., 2005) and 4F5 (Su et al., 2007), neither of which are commercially available. Moreover, although useful in cross- linking and labeling surface receptor, these antibodies may not indicate where receptors are sorted to after internalization due to the possibility of them dissociating from the receptors during endosomal maturation. These antibodies against the extracellular domains of FcγRII do not work well for immunofluorescence either. Alternatively, FcγRIIB can be preferentially engaged with immune complexes after other FcγRs are blocked (e.g. FcγRIIA with Fab fragments of IV.3, FcγRI with monomeric IgG). One caveat that should be taken into consideration is that specific engagement of some types of FcγRs can affect the activation/maturation status of primary myeloid cells in addition to triggering receptor- mediated endocytosis. For example, ligation of FcγRIIA or selective blockade of FcγRIIB promotes maturation of human DCs (Boruchov et al., 2005; Dhodapkar et al., 2005) and may result in the alteration of the trafficking pattern of FcγRs and immune complexes.

The third challenge lies in the lack of antibodies that specifically detect the divergent

FcγRIIA and FcγRIIB2 intracellular domains for use in immunofluorescence, which may be the reason why the subcellular localization of these receptors has not been investigated to date.

Data presented in chapter 3 suggests that antibody complexes internalized by FcγRIIA are sorted into lysosomal compartments in all three types of primary human myeloid cells.

However, as the sorting of FcγRs may diverge from that of their ligand, it is important to directly examine the trafficking of receptors with antibodies specifically against the intracellular domains of FcγRIIA or FcγRIIB. These antibodies need to work well for immunofluorescence as the number of monocytes isolated per blood draw is not enough for performing immunoblotting. Due to the high background staining of a commercially available

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goat anti-FcγRIIA antibody (Santa Cruz), I attempted to develop my own antibodies in rabbit.

Although the generated antibody does detect FcγRIIA in FcγRIIA-expressing ts20, the non- specific background staining still remains high (Fig. 3-5A). A prominent cross-reacting band at

36kDa was detected by FcγRIIA-specific antibody (i.e. recognition of the band could be depleted by preincubating serum with GST-FcγRIIA protein) and thus cannot be removed by affinity purification (Fig. 3-5B). My second attempt to generate an antibody through the services of a company was unsuccessful, suggesting the technical difficulty in generating such anti-FcγRIIA antibody. To generate anti-FcγRIIA antibodies with better specificity, a smaller region of FcγRIIA cytoplasmic tail instead of the entire intracellular domain may be used.

5.2.3 Future Plan

Further studies will be required to compare the internalization and the intracellular sorting of FcγRIIA and FcγRIIB2 upon engagement for endocytosis in human monocytes as well as different types of ex vivo differentiated macrophages and DCs. The regulation of FcγRIIA and

FcγRIIB expression can be investigated first in myeloid-lineage cells at a range of states of differentiation and activation. These experiments will help us understand how various FcγRII- mediated functions are modulated in different myeloid cells.

To examine FcγRIIA and FcγRIIB2 trafficking upon endocytosis, these two receptors can be either specifically engaged or co-engaged with anti-FcγRIIA IV.3, anti-FcγRIIB 4F5 or pan- anti- FcγRII AT10 antibodies. Alternatively, FcγRIIA- or FcγRIIB2-mediated endocytosis may be triggered by immune complexes following blocking pre-treatment with Fab fragment of 4F5 or IV.3 respectively. Findings from the transfected cell model shown in this thesis will be recapitulated in primary human myeloid cell system. Some aspects of FcγRII trafficking that can be investigated include:

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1) The kinetics of endocytosis mediated by FcγRIIA or FcγRIIB2. Requirements of the induction of endocytosis (i.e. receptor phosphorylation, ubiquitylation) by inhibitor pre- treatment and immunofluoresence.

2) The fate of the receptors by immunofluoresence and flow cytometry using anti-

FcγRIIA or anti-FcγRIIB2 antibodies.

3) The sorting of these receptors into various endosomal compartments by immunofluorescence co-staining with different endosomal markers. Immuno-electron microscopy approach will reveal more clearly the specific endosomal compartments FcγRIIA and FcγRIIB2 are transported into upon specific engagement or co-engagement (e.g. the trafficking of FcγRIIA into Hrs-positive MVB by microscopy).

Another important aspect of these studies is to investigate whether FcγRII transport in ex vivo differentiated human myeloid cells indeed reflect what happens in in vivo myeloid cells.

Among the various DC and macrophage subsets, the ones that circulate in blood (e.g. CD11c+ blood myeloid DCs) are accessible for experiments by staining with cell lineage markers and fluorescence activated cell sorting (FACS).

5.3 Identification of E3 ligase and ubiquitylation sites for FcγRIIA ubiquitylation

It has been demonstrated by the Booth lab that ubiquitin-conjugating system is essential for FcγRIIA endocytosis into clathrin-coated pits (Booth et al., 2002; Mero et al., 2006). The receptor itself becomes ubiquitylated upon clustering with multivalent immune complexes (Fig.

4-4).

Cbl is one of the E3 ligases commonly involved in ubiquitylation-dependent trafficking of receptors such as EGFR (Madshus et al., 2009), TCR (Naramura et al., 2002) and FcεR (Paolini

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et al., 2002). Mammalian members of the Cbl family include c-Cbl, Cbl-b and Cbl-3 (Blake et al., 1991; Keane et al., 1999; Keane et al., 1995). These proteins share a tyrosine kinase binding (TKB) domain that is associated with a Really Interesting New Gene (RING) finger domain through a linker (Thien and Langdon, 2005). The RING finger domain plays a key role in mediating the transfer of ubiquitin from E2 ubiquitin-conjugating enzyme to the target receptor. c-Cbl and Cbl-b also contain a proline-rich region and a ubiquitin-associated domain in their C-terminus. The ubiquitin-associated domain allows for the interaction of Cbl molecules with each other or other proteins containing ubiquitin or ubiquitin-like domain.

Cbl has been implicated in both the endocytosis and lysosomal targeting of EGFR

(Madshus et al., 2009). Cbl-mediated EGFR ubiquitylation is one of the several mechanisms of clathrin-dependent EGFR endocytosis that function in redundant and cooperative fashion (Goh et al., 2010). Combined knockdown of c-Cbl and Cbl-b expression by siRNA or overexpression of dominant negative Cbl results in inhibition in EGFR endocytosis (Huang et al., 2007; Huang et al., 2006a; Jiang and Sorkin, 2003). Cbl directly interacts with EGFR and mediates a sustained receptor polyubiquitylation after internalization (Grovdal et al., 2004;

Umebayashi et al., 2008). This process is essential to the delivery of EGFR to the limiting membrane of Hrs-positive MVB and its eventual degradation (Duan et al., 2003; Longva et al.,

2002).

c-Cbl has been implicated in FcγRIIA degradation in human neutrophils (Marois et al.,

2009). However, this loss of FcγRIIA in neutrophils which occurs very rapidly and requires proteasome activity may be a mechanistically unique feature of these cells. It is thus worthwhile to investigate the E3 ligase responsible for FcγRIIA internalization and sorting in other human myeloid cells. Some preliminary results by our group show the co-localization of

FcγRIIA and Cbl-GFP on the cell surface upon receptor clustering in ts20 transfected cells

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(data not shown). For future study, dominant negative Cbl without a functional RING finger domain may be utilized to determine if Cbl is required for FcγRIIA internalization and intracellular sorting. Alternatively, such a loss-of-function assay may be carried out by siRNA- mediated knockdown of Cbl expression level. If Cbl is not the E3 ligase responsible for ubiquitylating FcγRIIA during endocytosis, an shRNA screen for other candidate molecules may be attempted, focusing especially on those that are known to be involved in the ubiquitylation of other membrane receptors.

FcγRIIA-mediated endocytosis is ubiquitylation-dependent (Fig. 4-2), but none of the five lysine residues in the intracellular domain of FcγRIIA are essential for this process (Fig. 3-

21). It is possible that FcγRIIA is ubiquitylated at non-lysine residues in its intracellular domain upon agIgG engagement. To test this hypothesis, the potential non-lysine ubiquitylation sites in FcγRIIA including cysteines, serines and threonines can be mutated and the ubiquitylation of these FcγRIIA mutants will be examined.

5.4 Association of FcγRIIA trafficking and signaling

The signaling and trafficking of FcγRIIA, both triggered by ligand binding on the cell surface, have been extensively studied. However, the relationship between these two processes remains unclear.

There are several ways by which membrane receptor transport may contribute to the regulation of receptor signalling. Although the signalling cascade is generally thought to be initiated at the plasma membrane, there has been increasing evidence that in some cases signalling can also take place on endosomes after internalization (Murphy et al., 2009). One way to shut down receptor signalling is through the removal of receptors from the cell surface into endosomal compartments. Signalling by receptors such as EGFR can also be stopped

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through intracellular sorting of ligand-receptor complexes into lysosomal compartments

(Madshus et al., 2009). Receptors become segregated from kinases as they are transferred from the limiting membrane into the inner vesicles of MVB, resulting in signalling termination. The subsequent lysosomal degradation of receptors prevents further receptor signalling. This is in contrast to receptors that are recycled back to cell surface where they can be re-engaged with ligands to trigger signalling. One example of the receptors that undergo such a signalling termination process is TCR. Mice with double knockout of c-Cbl and Cbl-b in T cells exhibit a severe autoimmune phenotype due to sustained TCR signalling (Naramura et al., 2002). This is caused by reduced sorting of internalized TCR for lysosomal degradation resulting in decreases in TCR down-regulation after ligand engagement.

Our lab has recently shown that soluble agIgG induces transient signalling during which

Erk1/2 phosphorylation peaks at 15 minutes in primary human monocytes whereas surface- bound IgG elicits sustained signalling for hours (Gupta and Booth, 2010). Treatment of cells with the endosomal maturation inhibitor bafilomycin increases persistence of Erk1/2 phosphorylation in response to agIgG. This suggests that signalling termination following agIgG internalization in monocytes is likely to be mediated by sorting of receptors into multivesicular late endosomes instead of receptor removal from cell surface. However, human monocytes express both FcγRI and FcγRIIA that can be engaged or co-engaged by agIgG to trigger downstream signalling (Boruchov et al., 2005). Thus, it still remains unclear how

FcγRIIA trafficking contributes to its signalling termination. One possibility is that FcγRIIA signaling is turned off by its degradation or sorting into inner vesicles of MVB inaccessible to signaling molecules. To approach this, one may pre-treat monocytes with monomeric IgG to block FcγRI before testing the effect of bafilomycin on the phosphorylation of receptor or

Erk1/2 after agIgG uptake by FcγRIIA. Other approaches include interventions to limit

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FcγRIIA sorting to MVB such as siRNA knockdown of Hrs or other molecules important for targeting proteins to MVB. However, this cannot rule out the possibility that FcγRIIA signaling is stopped by removal of FcγRIIA from cell surface. This possibility may be tested by preventing FcγRIIA-mediated internalization of immune complexes such as inhibition with proteasome inhibitor or knocking down molecules cruicial to the induction of endocytic pathway such as clathrin.

5.5 Effect of Trafficking of FcγRIIA and FcγRIIB2 on Antigen Presentation

FcγR-mediated endocytosis can increase the efficiency of the presentation of antigen- derived peptides on the surface of APCs (Amigorena et al., 1999), although the mechanism underlying this is not fully clear. It has been shown in a mouse model that FcγRIIB2 endocytoses immune complexes at a similar rate as activating FcγRs (I, III, IV) but sorts immune complexes to different endosomal compartments (Bergtold et al., 2005; Desai et al.,

2007). It inhibits antigenic processing for MHC class-I mediated cross-presentation to activate

CD8+ T cells by competing with activating FcγR for limiting amount of internalized immune complexes. Evidence in the human system suggests an inhibitory role of FcγRIIB in activation of autologous T cells by immune complexes-stimulated DCs via FcγRIIA (Guriec et al., 2006).

However, the mechanism of such inhibition remains unclear. One possibility is that the ITIM of FcγRIIB inhibits FcγRIIA-mediated global effects on the cell leading to decreased antigen presentation. Alternatively, FcγRIIB2 may compete with FcγRIIA for internalized immune complexes and lead to trafficking of immune complexes to different intracellular compartments.

It is also not known how FcγRIIA-mediated endocytosis of immune complexes and trafficking contributes to MHC class-I/-II dependent antigen presentation.

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Future studies will focus on the implications of FcγRIIA and FcγRIIB2 trafficking in

MHC class-I and –II mediated presentations and the mechanisms that underlie such linkages.

The hypothesis will be that, in human DCs, the engagement of FcγRIIA leads to its trafficking into MIIC and loading of antigen-derived peptide onto MHC class-II, contributing to the enhancement of antigen presentation. Some antigens internalized via FcγRIIA can also exit endosomes and enter the MHC class-I-mediated presentation pathway, resulting in antigen cross-presentation to CD8+ T cells. In such case, the engagement of FcγRIIB2 results in a reduction in antigen presentation on both MHC class- I and -II by diverting immune complexes away from both processing pathways.

To investigate promotion of MHC class-II mediated antigen presentation by FcγRIIA or

FcγRIIB2, moDCs can be loaded with immune complexes after pre-blocking with IV.3 or 4F5 antibodies followed by examining their abilities to activate MHC class-II restricted T cells. A similar approach can be taken to study the association of MHC class-I antigen presentation and

FcγRII-mediated endocytosis using MHC class-I restricted T cells instead. The delivery of

FcγRIIA to MHC class-II containing MVB upon engagement with IV.3 antibody complexes may be analyzed by immunoEM in combination with anti-MHC class-II antibody. Another aspect of FcγRII-mediated antigen presentation is the effects of varying the ratio of expressions of FcγRIIA and FcγRIIB by cytokine treatments on antigen presentation in myeloid cells such as moDCs. The overall internalization of immune complexes under different conditions may be compared and whether an increase in FcγRIIA/FcγRIIB ratio increases presentation on both

MHC class-I and class-II will be examined.

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5.6 Clinical implications

Administration of high dose IVIg has been an effective therapy in treating a number of autoimmune diseases (Imbach et al., 1981; Schmidt et al., 1981). The best studied disease model is idiopathic thrombocytopenic purpura (ITP), a disorder involving the clearance of platelets mediated by pathogenic platelet-specific antibodies. Several mechanisms by which

IVIg exerts its therapeutic benefit have been proposed, with FcγRs as the key players. The protective effect of IVIg has been attributed to the blockage of activating FcγR activities by monomeric IgG in IVIG, leading to restoration of the overall signalling balance (Clarkson et al.,

1986; Debre et al., 1993; Schmidt and Gessner, 2005). This is consistent with the requirement for FcγRIIB of ITP amelioration by IVIg and the increase in FcγRIIB expression on macrophages upon IVIg treatment (Crow et al., 2003; Samuelsson et al., 2001).

Small immune complexes (e.g. IgG dimers) representing a small fraction of IVIg have also been shown to play an active role in controlling platelet clearance in ITP (Boughton et al.,

1990). These small-sized immune complexes and dimer-enriched IVIg are more effective in ameliorating ITP (Bazin et al., 2004; Siragam et al., 2006; Teeling et al., 2001). Lazarus and his colleagues suggested that IVIg interacts directly with the activating FcγRs on DCs which subsequently up-regulate macrophage FcγRIIB, leading to an inhibition in platelet clearance

(Crow et al., 2009). This is consistent with the finding that surface expression of FcγRIIA but not FcγRIIB is downregulated in human immature moDCs when treated with IVIg in vitro

(Boruchov et al., 2005). Based on my findings, it is possible that the small-size immune complexes in IVIg exert an anti-inflammatory effect through co-engagement of FcγRIIA and

FcγRIIB which leads to accelerated downregulation of the overall FcγRIIA level and damping of activating signalling. IVIg treatment has been wildly used for a range of autoimmune diseases, but it is generally ineffective in patients with rheumatoid arthritis and has only

132

marginal benefit for concurrent SLE (Jolles et al., 2005; Knezevic-Maramica and Kruskall,

2003). An alternative approach to IVIg will be to replace high-dose IVIg with a low-dose of humanized monoclonal antibodies such as AT10 antibodies that may coengage FcγRIIA and

FcγRIIB leading to downregulation of activating signals and inflammatory responses as explained in Fig. 5-2.

Another application of my study lies in vaccine design. Antigen internalized through

FcγRs as part of the immune complexes are presented more efficiently after processing on the surface of APCs to activate both CD4+ and CD8+ T cells than those taken up in fluid phase.

Antigen-containing immune complexes may be used in vaccine design to enhance vaccine efficacy, especially for antiviral vaccines where purified pathogen-derived protein subunits are commonly used. Different FcγRs vary in their sorting route and their capabilities of mediating antigen presentation. To further enhance vaccine efficacy, one may incorporate antibodies into the new vaccine that specifically target a class of FcγRs that are best at promoting antigen presentation through accessing MIIC. This property of FcγRs may also be utilized in improving DC immunotherapy where the presentation of pathogen-specific or cancer-derived antigens can be enhanced through FcγR-mediated endocytosis of antigen-containing immune complexes.

5.7 Conclusion

The findings presented in this thesis show that there are fundamental differences in the trafficking behaviour of different FcγRs, in particular FcγRIIA and FcγRIIB2. When engaged with immune complexes, FcγRIIA is sorted into lysosomes for degradation whereas FcγRIIB2 accesses recycling endosomes for trafficking back to the cell surface. Upon co-engagement, immune complexes dissociate from the two receptors prior to the sorting of these receptors to

133

their distinct final fates. FcγRIIB2-mediated endocytosis, unlike that of FcγRIIA, is not ubiquitylation-dependent and occurs at a faster rate. This study provides insights into how antigens in immune complexes internalized via potentially multiple FcγR subtypes are handled by human myeloid cells.

Human and mouse FcγR families differ in many aspects including the presence distinct members (human: FcγRIIA; mouse: FcγRIV) and binding affinities of to IgG subclasses. Much work has been done to study the signalling and trafficking of mouse FcγRs, but our understanding in the biology of human FcγRs is still limited. Given the restricted presence of

FcγRIIA to primates and its known polymorphism H/R131 associated with autoimmunity, the studies presented here are particularly relevant in designing new vaccines or novel therapeutic strategies for controling the manifestation of human antibody-mediated autoimmune diseases.

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