CD8 t cell mediated induction of interleukin 12p70 production by dendritic cells

Though previous studies had shown that CD8 T cells had a direct influence on DC IL-12p70 production, they were not without their limitations.. In this study, we set up an in vitro system

CONTROL OF DENDRITIC CELL INTERLEUKIN-12

Summary

IL-12 is the central cytokine in the initiation and maintenance of a Th1 immune response

The production of IL-12p70 by DCs during T cell priming is crucial for the development

of Th1 responses Hence, control of DC IL-12p70 production is key for the development

of Th1 immunity DC activity can be influenced through direct and indirect interaction

with other effector immune cells This results in the delivery of “feedback” signals that can

influence the activation status of the DC and thus the outcome of the adaptive immune

response While CD8 T cells are primarily known as cytotoxic T cells, their ability to

modulate immune responses through interactive feedback with DCs has also been

described

Of particular interest in this study was the ability of CD8 T cells to promote the production

of IL-12 during their interaction with DCs Though previous studies had shown that CD8

T cells had a direct influence on DC IL-12p70 production, they were not without their

limitations First, all the DCs utilized so far were generated from DC precursors using

GM-CSF and IL-4 It is now known that these DCs do not represent lymphoid resident

DCs, which consist of a heterogeneous mix of DC subtypes The ability of CD8 T cells to

modulate IL-12p70 production with these DCs is not known

Second, while IFN-γ was found to be the main factor involved in DC IL-12p70 priming,

other studies suggests that this could occur through additional mechanisms It is also

unclear if the immune status of CD8 T cells, i.e nạve, activated or memory was important

for the ability of CD8 T cells to prime DCs for IL-12p70 Finally, it is uncertain in what

immunological context CD8 T cells could influence DCs for IL-12p70 production

In this study, we set up an in vitro system to study the effects of CD8 T cells on splenic

DC IL-12p70 production during an antigen specific interaction We find that mouse

splenic DCs respond to CD8 T cells with IL-12p70 production only when DCs were

concurrently stimulated with TLR ligands Different TLR ligands cooperated with CD8 T

cells for IL-12p70 production to different extents In particular, CpG was most effective

for the induction of IL-12p70, while other TLR ligands, like LPS, were only moderately

effective We also found the immune status of the CD8 T cells was important for priming

DCs for IL-12p70 production Pre-activation of CD8 T cells was required for effective DC

IL-12p70 priming with LPS With CpG, naturally occurring memory CD8 T cells were

superior to nạve CD8 T cells for the induction of IL-12p70 Furthermore, we also show

that apart from IFN-γ, CD8 T cells also use CD40L, GM-CSF and TNF-α to prime DCs

for IL-12p70 production Of the splenic DC subsets, CD8α+ DCs were the primary source

of IL-12p70, demonstrating the specialized role of the CD8α+ DC subset in this process

Interestingly, CD8α+ DCs are also implicated as the principle DC subset for

cross-presentation and cross-priming cytotoxic CD8 T cell responses Our study describes a

novel pathway for the induction effective IL-12p70 responses to cross-presented antigens

Overall, this study showed that CD8 T cells have the capacity to modulate DC IL-12p70 in

the presence of microbial stimuli that triggers DC TLRs It is possible that during the

course of an infection, activated or memory antigen specific CD8 T cells could provide

essential stimuli to DCs for the production of IL-12p70 that result in protective Th1

responses The current study furthers the understanding of how CD8 T cells can influence

DCs, opens up new avenues of research and is useful for the development of therapeutics

that seek to promote cell mediated immunity or suppress allergic immune responses

Acknowledgements

Being able to write this acknowledgment means so much to me It means I am at the end

of the road to fulfilling my childhood dream of finally becoming a “doctor” Someone once told me a PhD is your passport to the world, the real beginning as a scientist But for every new beginning, there is an end It was a journey that I shared with so many wonderful people that are now my dearest friends

To Prof Kemeny, I will always remember the day when I first approached you Given the desperate circumstances, I am etherally grateful for the fact that you did not turn me down

I am also grateful for the opportunities you had given me, and the freedom to pursue my own research interests Though I know I don’t live up to your every expectation, your supervision has made me into a much stronger, tougher person that is now ready to face the world

To Dr Paul, thanks for being there for me when I needed help the most, especially for teaching me experimental techniques, the fine art of manuscript writing and invaluable advice on how to be a scientist and to do science You had certainly played a big role And you know what, we are both new fathers! To Dr Veronique, though we only communicated a few times, I felt that I learnt a lot each from each communication Thanks for your stories of inspiration and perseverance that kept me going during tough times, and thanks for trusting me and my judgement Here’s wishing you many many wonderful years

of fulfilling research and family life

To Shuzhen, thanks for the times we shared Knowing you has been a life changing experience for me Thank you for vetting my thesis, which is one “small” thing out of so much that you had done To Fei Chui, it is always inspiring to see the fighter that you are Thanks also for all the motherly care you had given, which reminds me on how a mother hen take cares of her hatchlings And finally, a real hatchling is coming! To Issac, thanks for being such a great buddy and activity partner! From soccer, playing computer games to eating at BJ, what else could a buddy ask for? So what now? To Adrian, thanks you for always being so honest and skeptical about almost everything Though you have strong opinions about things, you almost always end you sabotaging people when they listen to you I find that very hilarious and that is the most endearing quality about you By the way, welcome to fatherhood! To Yafang, thanks for listening to me talk, complain and worry about things, you are so adorable and with your shoes that ring and everything Your sky high IQ and streetwise-ness that make me “so scared” of you, because I think that you know things that I don’t know you know! To Zhenying, you seem to be so innocent and honest, but yet I know you enjoy it most when you crack the most awarkward of jokes just

to make me flabbergasted! I really enjoyed your company and thanks for hearing out me out when I am having problems *Wink* To Kenneth and Richard, thanks for tolerating

my “bad attitude” towards you and my constant barrage of rude remarks It was good fun and sometimes you just need a punching bag in life, which happens to be you guys Kenneth, you are one of the nicest persons I had ever met, always thinking of others and putting them first over yourself Could you be more human and show your selfish side once a while? To Melissa, you are everything that a co-worker would ask for Though I

find the word “obedience” awkward, it was your greatest asset to me You had certainly had come a long way and perhaps it is a good time for you to find your independence To Pey Yng and Chris, thanks for continuing the work and taking a special interest in the molecular side of things To Javier, thanks for being such an easy going person and lending me your computer games but without the CD keys To Benson and Elsie, thanks for keeping the lab running smoothly I would also like to thank other friends including Angeline, Karwai, Xilei, Lin Gen, Jason, Boon King, Olivia and others not mentioned who had made my life so much more interesting

Special thanks goes to Jane Thanks for enduring so much for me and tolerating all my nonsense that I throw at you when I am fustrated Thanks for always being there, always finally listening to me and for taking care of Kaizer, which is no small task To Kaizer, thanks for making my life such a mess, but without which I wouldn’t have known the true value of love, life and God Do grow up to be a virtuous man Thanks to Jane’s family, Richard and Lily, though I know you hate it when I address you by your names Your care, help and support was and is still invaluable To mum and dad, thanks for brining me up so that I could see this day, and my bother, may you live a happy and fulfilling life

MACS Magnetic activated cell sorting

Table of contents

Chapter 1: Introduction 17

Interleukin-12 17

CD8 T cells 18

Dendritic cells 20

Heterogeneity of DCs 21

T helper responses: the different flavors 23

Polarization of the immune response by DCs 26

Toll-like receptors 28

TLR stimulation and the development of Th1 and Th2 polarizing DCs 30

Propensity of different DC subsets to induce Th1 and Th2 responses 32

Modulation of DCs by interacting cells 33

Modulation of DCs by CD8 T cells 35

Aims of this study 37

Specific aims 38

Chapter 2: Materials and methods 39

Media and buffers 39

PBS buffer 39

MACS/FACS buffer 39

Permeabilization/washing buffer for intracellular staining 40

Annexin-V binding buffer 40

Optiprep density centrifugation media for splenic DC isolation 40

Digestion buffer for spleen DC isolation 41

Complete RPMI for cell culture 41

Buffers for ELISA 41

Mice 42

Cell isolation 42

Splenic DC isolation 42

Splenic CD8 T cell isolation 45

Preparation of cells for FACS 48

Surface staining of cells for FACS analysis 48

Intracellular staining of cells for FACS analysis 49

Preparation of cells for sorting 50

CD8 T cell DC co-cultures 51

Detection of cytokines in supernatants 52

ELISA 52

Multiplex bead array 53

CTL killing 55

51 Cr release assay 55

Annexin-V and 7-AAD viability assay 56

Statisical analysis 56

Chapter 3: Requirements for CD8 T cell mediated DC IL-12p70 production 61

Introduction 61

Results 62

Purity of MACS isolated splenic CD8 T cells and DCs 62

CD8 T cells up-regulate the surface expression of co-stimulatory molecules on

Pre-activation of CD8 T cells was required for the induction of DC IL-12p70 66

LPS or endotoxin was required for CD8 T cell mediated IL-12p70 by DCs 68

Kinetics of activated CD8 T cell mediated IL-12p70 production 72

IL-12 is produced by DCs during co-culture 74

Priming of DC for IL-12p70 production was Kb/OVA257-264 specific 76

DCs were not killed by CD8 T cells during an antigen specific interaction 79

Multiplex analysis of cytokines and chemokines produced during co-culture 83

Discussion 87

Chapter 4: Mechanisms of CD8 T cell mediated IL-12p70 production 91

Introduction 91

Results 93

Soluble factors involved in the priming of DCs for IL-12p70 production 93

CD8 T cell mediated IL-12p70 production is contact dependent 95

CD40-CD40L interaction is essential for CD8 T cell mediated IL-12p70 production 97

The effects of blocking IFN-γ and CD40L on IL-12p70 levels over time 99

CD40L is rapidly up-regulated by CD8 T cells 101

CD8 T cells pre-activated by anti-CD3 and anti-CD28 or OVA257-264 peptide were unable to provide sufficient CD40L stimulation to DCs for IL-12p70 production 103

The CD8α+ DC subset is responsible for CD8 T cell mediated IL-12p70 production 105

Proportion of DC subsets in CD40-/- and WT DCs are similar 109

CD40-/- DCs are not inherently defective for IL-12p70 production 111

Discussion 113

Chapter 5: Cooperation of CpG with CD8 T cells for DC IL-12p70 priming 116

Introduction 116

Results 118

TLR ligands induce splenic DCs to up-regulate co-stimulatory molecules in a dose dependent and saturable manner 118

CpG most potently cooperates with antigen specific CD8 T cells for the induction of DC IL-12p70 production 120

Isolation of pDCs and cDCs from CD11c MACS cell preparations 126

Activation of pDCs by CpG and CD8 T cells 129

Contribution of pDCs and cDCs to the IL-12p70 response 132

Contribution of CD8α+ and CD8α- cDC subsets for IL-12p70 production 133

Discussion 135

Chapter 6: Memory CD8 T cells are superior in their ability to prime CpG stimulated DCs for IL-12p70 138

Introduction 138

Results 139

Role of soluble factors in CpG/CD8 T cell mediated IL-12p70 production 139

CD40-CD40L interactions in CpG/CD8 T cell mediated IL-12p70 production 141

Differential expression of surface markers by CD44hi memory phenotype and CD44lo nạve phenotype CD8 T cells 145

CD44hi memory phenotype CD8 T cells are superior in their ability to prime DCs for IL-12p70 147

CD44hi and CD44lo CD8 T cells express similar levels of CD40L during co-cultures 149

CD44hi CD8 T cells secrete more IFN-γ and GM-CSF during co-cultures that

Discussion 153

Chapter 7: Final discussion 156

Summary 156

The activation state of CD8 T cells correlates with its ability to prime DC IL-12p70 156

The superior ability of memory CD8 T cells for IL-12p70 priming ability 159

DC subsets in CD8 T cell mediated IL-12p70 production 161

The necessity of TLR stimulation 162

The possible role of direct stimulation of CD8 T cells by TLR ligands 164

CD8 T cell mediated IL-12p70 production in humans 166

Limitations of current study 167

References 169

List of Tables

Table 2.1 Antibodies used in this study 58

Table 2.2 Reagents used in this study 59

Table 3.1 23-plex bead array for cytokine and chemokine levels found in co-cultures of freshly isolated and activated CD8 T cells 84

List of Figures Fig 1.1 Known Mouse DC subsets and their major characteristics 24

Figure 1.2 DCs integrate signals from various sources and differentiate into Th1 or Th2 inducing DCs 27

Fig 1.3 Cellular locations of TLRs and their respective ligands 29

Fig 3.1 FACS profile of CD8 T cells and DCs after positive selection with MACS 63

Fig 3.2 CD8 T cells induce the up-regulation of co-stimulatory molecules on splenic DCs during a peptide specific interaction 65

Fig 3.3 IL-12p70 levels during co-culture of CD8 T cells and OVA257-264 pulsed DCs 67

Fig 3.4 Endotoxin levels found various batches of FCS and other reagents used 69

Figure 3.5 Activated CD8 T cells effectively prime splenic DCs for IL-12p70 production 71

Fig 3.6 Kinetics of IL-12p70 production 73

Fig 3.7 Intracellular staining for IL-12p40 75

Figure 3.8 CD8 T cell mediated IL-12p70 production is MHC class I-peptide dependent 78

Figure 3.9 Viability of DCs after co-culture with activated CD8 T cells 81

Figure 3.10 CD8 T cell killing of peptide pulsed EL4 82

Figure 4.1 Role of cytokines in CD8 T cell mediated IL-12p70 production 94

Figure 4.5 CD40L expression by activated CD8 T cells 102

Figure 4.6 IFN-γ and CD40L expression profile of differentially activated

CD8 T cells after co-culture 104

Figure 4.7 FACS profile of sorted DCs and IL-12p70 levels after co-culture of

sorted DCs 107

Figure 4.8 The splenic CD8α+ DC subset is responsible for IL-12p70 production 108

Figure 4.9 Proportion of DC subsets in WT and CD40-/- DCs 110

Figure 4.10 Production of IL-12p70 by WT and CD40-/- DCs in response to IFN-γ

and CpG 112

Figure 5.1 Up-regulation of co-stimulatory molecules after treatment of DCs with

different TLR ligands 119

Figure 5.2 Levels of IL-12p40 and IL-12p70 during co-culture of freshly isolated

CD8 T cells and DCs together with various TLR ligands 123

Figure 5.3 The effect of different concentrations of CpG on IL-12p70

production induced by freshly isolated CD8 T cells 124

Figure 5.4 Levels of IL-12p70 during co-culture of activated CD8 T cells and DCs

together with various TLR ligands 125

Figure 5.5 Differential expression of surface markers by pDCs and cDCs 127

Figure 5.6 Percentage of pDCs increases with longer incubation times with

CD11c MACS beads 128

Figure 5.7 FACS sorting of splenic pDCs and cDCs 130

Figure 5.8 Activation of pDCs by CD8 T cells and CpG 131

Figure 5.9 Isolation of cDC and pDC by FACS and production of IL-12p70 by

sorted DCs during co-culture 132

Figure 5.10 IL-12p70 levels with sorted DCs 134

Figure 6.1 Role of IFN-γ, TNF-α and GM-CSF in nạve CD8 T cell mediated

Fig 6.5 Differential induction of IL-12p70 by CD44hi and CD44lo CD8 T cells 148

Fig 6.6 Expression of CD40L by CD44hi and CD44lo CD8 T cells 150

Fig 6.7 Differential production of IFN-γ, GM-CSF and TNF-α by CD44hi and

CD44lo CD8 T cells 152

Fig 7.1 Schematic representation DC IL-12p70 priming by CD8 T cells 158

IL-12p70 production (Under review, Journal of Immunology)

CD8 T cells induce human monocyte derived dendritic cells to produce IL-12p70 in the presense of TLR4 activation (Mauuscript in preparation)

Chapter 1:

Introduction

Interleukin-12

IL-12p70 is a heterodimeric pro-inflammatory cytokine that plays a central role in Th1

immunity (Trinchieri, 2003) It is produced by APCs, such as dendritic cells, monocytes

and macrophages IL-12p70 primes T cells and NK cells for the production of high levels

of IFN-γ and activates the cytotoxic function of CD8 T cells and NK cells During T cell

priming, IL-12p70 is the primary factor produced by DCs that drives development of Th1

responses (Heufler et al., 1996; Macatonia et al., 1995; Trinchieri, 2003) IL-12 is also

required to maintain ongoing Th1 responses (Park et al., 2000; Yap et al., 2000) The

IL-12 receptor is composed of two chains, the IL-IL-12Rβ1 and IL-IL-12Rβ2 The IL-IL-12 receptor is

expressed by activated T cells and NK cells (Presky et al., 1996), DCs and B cell lines

(Airoldi et al., 2000; Grohmann et al., 1998) CD4, Th1 cells but not Th2 cells, express

IL-12Rβ2 and respond to IL-12 (Rogge et al., 1997; Szabo et al., 1997)

IL-12 is a heterodimeric cytokine consisting of covalently linked p35 and p40 subunits

The genes encoding the p35 and p40 are located on separate chromosomes, chromosome 3

and 5 in humans, and chromosome 6 and 11 in mice (Trinchieri, 1998) While p40

homodimers are usually produced in large excess over the p70 heterodimer, the Th1

inducing capacity is restricted to the IL-12p70 heterodimer (Gubler et al., 1991; Kobayashi

been shown to bind to IL-12Rβ1 which inhibits the activity of IL-12p70

(Gately et al., 1996; Gillessen et al., 1995; Mattner et al., 1993), it also acts as a

chemoattractant to macrophages (Ha et al., 1999) and induces DC migration (Khader et

al., 2006) Differential control of the transcription of p35 and p40 subunit underlies the

observation that a single stimulus results in modest IL-12p40 but not IL-12p70

production For example, only stimulation of DCs with LPS together with IFN-γ, but not

with LPS or IFN-γ alone, results in the production of IL-12p70 (Snijders et al., 1998)

The importance of IL-12 is exemplified by the immune phenotypes of IL-12 KO mice

(Hunter, 2005) IL-12p40 deficient mice are very susceptible to infection with several

intracellular pathogens IL-12p35 KO mice are deficient in Th1 responses, susceptible to

intracellular pathogens but to a lesser extent than 12p40 KO mice Furthermore,

IL-12p35 deficient mice but not IL-12p40 deficient mice developed experimental

autoimmune encephalomyelitis (Becher et al., 2002; Gran et al., 2002) These

observations can be explained by the fact that the p40 subunit associates with the p19

subunit to form a different heterodimeric cytokine with distinct functions, IL-23

(Oppmann et al., 2000) IL-23 promotesthe differentiation of nạve CD4 T cells to Th17

cells and induces proliferation of memory T cells (Park et al., 2005) It is also involved in

the development of inflammatory and autoimmune associated diseases (Cua et al., 2003;

Langrish et al., 2005; Murphy et al., 2003; Wiekowski et al., 2001)

CD8 T cells

CD8 T cells are known as cytotoxic T cells that recognize antigenic peptides presented by

MHC class I All nucleated cells express MHC class I, but only infected or tumor cells

present the appropriate antigenic peptides on MHC class I that makes them targets for

recognition and susceptibile to killing by CD8 T cells CD8 T cells are crucial for the

elimination of infected and tumor cells

CD8 T cells start off as nạve T cells that circulate the body after development in the

thymus CD8 T cells are activated by encounter with antigen, which results in their

proliferation and differentiation into effector T cells Activated CD8 T cells express

molecules associated with killing, like perforin and granzyme, and also high levels of

Th1 associated cytokines, like IFN-γ While CD8 T cells typically produce large amounts

of IFN-γ upon activation, the differentiation of Tc2 CD8 T cells that secrete IL-4 can be

generated in vitro (Le Gros & Erard, 1994; Sad et al., 1995) and are associated with

several immunological pathologies (Inaoki et al., 2003; Ito et al., 1999; Maggi et al., 1994;

Ying et al., 1997) has also been described

The ability of the immune system to mount a rapid and efficient response against a

previously encountered pathogen is conferred by “memory” antigen specific T and B

cells Memory lymphocytes arise as a consequence of encounter with foreign antigens

These lymphocytes acquire enhanced survival capabilities and are able to respond

proliferate and perform effector functions rapidly upon subsequent antigenic re-encounter

(Dutton et al., 1998; Dutton et al., 1999) Memory CD8 T cells are thought to develop

from the effector cell pool and are important for protection against recurrent infection by

intracellular pathogens

Dendritic cells

DCs play a central role in immunity They are vital for detecting, alerting and priming the

adaptive immune system to invading pathogens (Banchereau et al., 2000; Banchereau &

Steinman, 1998) According to the classical paradigm, DCs are strategically located in

peripheral tissues like the skin in an immature state where they can respond rapidly to

invading pathogens These immature DCs are immune sentinels, typified by their

extraordinary ability to sample their local environment for pathogens Immature DCs are

highly efficient at macropinocytosis and endocytosis (Guermonprez et al., 2002; Sallusto

et al., 1995) Once they encounter pathogens, they undergo activation and profound

changes, a process collectively termed “maturation”

Maturation transforms immature DCs from efficient antigen capturers into professional

antigen presenters This results in an almost complete down-regulation of antigen capture,

which is coupled with a dramatic increase in antigen processing ability (Steinman &

Swanson, 1995) The ability of DCs to process antigen is attributed to specialized

intracellular MHC class II-enriched compartments that are present only in DCs (Watts,

1997) Internalized antigens are directed to these compartments where they are degraded

and subsequently loaded onto MHC class II molecules MHC class II peptide complexes

are then directed to the surface for presentation to CD4 T cells Mature DCs also process

and present antigenic peptides loaded on MHC class I for priming CD8 T cells Maturing

DCs that have captured antigen subsequently migrate to the T cell region of secondary

lymphoid organs (Randolph et al., 2005) Mature DCs also increase the expression of

co-stimulatory molecules and extend characteristic “dendritic” extensions and membrane

folds that enable them to form close contact with multiple T cells simultaneously Mature

DCs also secrete cytokines that have a powerful impact on the outcome of the adaptive

immune response (Banchereau & Steinman, 1998)

Heterogeneity of DCs

While Langerhans cells and dermal DCs reside in the skin and migrate to lymph nodes

upon activation (Valladeau & Saeland, 2005), various non-migratory DCs exist in

lymphoid organs, where they deal with blood or lymph borne microbial threats (Henri et

al., 2001; Vremec et al., 2000) Lymphoid resident DCs are heterogeneous and have

distinct phenotypic and functional properties (Shortman & Liu, 2002) These different

DC subsets can be identified using CD4 and CD8α In the spleen, three distinct

populations of DCs can be identified They are CD4+CD8α-, CD4-CD8α+ or CD4-CD8α

-(Vremec et al., 2000) The proportion of these DCs is roughly 2:1:1 Skin draining lymph

-CD8α-CD11b+CD205mid dermal DCs and CD8αloCD11b+ CD205hi Langerin+ Langerhans

DCs (Anjuere et al., 1999; Henri et al., 2001)

Non-classical DCs subsets include the plasmacytoid DCs (Asselin-Paturel et al., 2001;

Grouard et al., 1997; Liu, 2005; Nakano et al., 2001) At resting state, pDCs resemble

plasma B cells by having an expanded cytoplasm and extensive endoplasmic reticulum

They express high levels of TLR7 and TLR9 and respond rapidly to viruses by the rapid

production of high levels of type I interferon This property makes them an important

component of the anti-viral response pDCs eventually mature and adopt DC morphology

B220+ Gr1(Ly-6C)+ DCs with NK cell like activity, called interferon-producing killer

DCs (IKDCs) has also been described (Chan et al., 2006; Taieb et al., 2006) These rare

cells kill cells that lack MHC class I, produce large amounts of type 1 interferons and

eventually mature into classical DCs that process and present antigens to T cells They

express markers for NK cells CD49b, NK1.1 and NKG2D, the plasmacytoid DC marker

B220 and also the classical DC marker, CD11c

Different subsets of DCs have specialized functions For instance, CD8α+ DCs are the

primary DC subset for cross-presentation and cross-priming cytotoxic CD8 T cell

responses, while CD8α- DCs preferentially primes CD4 T cells (Belz et al., 2005; Belz et

al., 2004a; den Haan & Bevan, 2002; den Haan et al., 2000; Pooley et al., 2001; Smith et

al., 2003) The cross-presentation and cross-priming ability of CD8α+ DCs is largely due

to the expression of cross-presentation machinery that is largely absent in other DC

subsets (Dudziak et al., 2007; Schnorrer et al., 2006) Different classical DC subsets react

differently to microbial stimuli and are innately predisposed to produce different

cytokines and chemokines (Hochrein et al., 2001; Proietto et al., 2004; Shortman & Liu,

2002) and to polarize T cells into different functional subsets (discussed later) Evidence

also suggests that migratory skin DCs can transfer antigens to lymphoid resident DCs that

prime T cells (Allan et al., 2006; Belz et al., 2004b; Inaba et al., 1998)

DCs are rare cells and hard to acquire in large numbers Hence, DCs are commonly

generated in vitro from human blood monocytes or mouse bone marrow progenitors

(Inaba et al., 1992; Sallusto & Lanzavecchia, 1994) These in vitro generated DCs had

been described as “inflammatory DCs”, or DCs that only appear as a consequence of

infection or inflammation (Naik et al., 2006; Shortman & Naik, 2007) These DCs do not

appropriately represent DCs at steady state, such as lymphoid resident DCs Hence,

results obtained from GM-CSF and IL-4 generated DCs might not properly translate to

events that can occur with lymphoid resident DCs Despite the heterogeneity of DCs,

DCs are defined by the common ability to prime nạve CD4 T cells CD11c is a pan-DC

marker in mice, while CD83 is a marker for activated DCs in humans MHC class II also

identifies all APCs, including DCs APCs also express co-stimulatory molecules like

CD80 and CD86 for activating T cells Fig 1.1 Illustratrates the different DC subsets that

can be found in the mouse

T helper responses: the different flavors

To date, at least four different CD4 T cell subsets that mediate different immune

responses have been described These are Th1, Th2, Th17 and Tregs (Harrington et al.,

2005; Langrish et al., 2005; Mosmann & Coffman, 1989; Park et al., 2005; Sakaguchi,

2004) Only Th1 and Th2 will be described here Th1 cells are characterized by their

ability to produce IFN-γ and TNF-β They are the main arm of cell mediated immunity

that is effective against intracellular bacteria, viruses and tumors Th2 cells produce IL-4,

IL-5, IL-13 that promotes humoral immunity effective against parasites Th2 cells are

also responsible for the development of asthma Generally, the response to a pathogen is

dominated by one T cell subset, and T cell subsets are antagonistic to each other This

underscores the importance of mounting a proper protective T helper response during an

Lymphoid tissue resident DCs

0on-classical DCs

Migratory skin resident DCs

CD4+CD8α+CD11b+33D1+IFN-γ production Preferentially primes CD4 T cells

CD4-CD8α+CD11b-DEC205+TLR7-Cross-presentation IL-12 production

CD4-CD8α+CD11b+Tolergenic?

Inflammatory DCs

CD11cloB220+ Gr1(Ly-6C)+mPDCA-1+High TLR9 and TLR7 High Type I IFN-α production Activation induces MHC class IIhi and

T cell priming activity

CD49b+NK1.1+NKG2D+B220+

CD11c+

NK cell activity Activation induces MHC class IIhi and T cell priming activity

Exist during inflammatory conditions Generated with GM-CSF + IL-4 in vitro Produces of high levels of inflammatory cytokines

CD8intCD11b+DEC205hiLangerin+Epidermis Tolerance?

Transfer of antigens to lymphoid resident DCs

CD4-CD8CD11b+CD205mid Transfer of antigens to lymphoid resident DCs

CD8α+

Langerhan cells

Dermal DCs

CD4

-CD8α

-Fig 1.1 Known Mouse DC subsets and their major characteristics

infection Furthermore, improper T helper responses results in persistent infection and

drives the onset of autoimmunity, atopy or allergy As DCs are the principle cell type that

prime T cells, controlling DC activity is the key to mounting a proper Th response

The differentiation of CD4 T cells into Th1 by DCs is largely mediated by IL-12p70

(Trinchieri, 2003) Additionally, the role of type I interferons (Brinkmann et al., 1993;

Parronchi et al., 1992), chemokines (Luther & Cyster, 2001) in Th1 polarization has also

been described IL-18 syngerzies with IL-12 for the induction of Th1 responses

(Nakanishi et al., 2001; Salagianni et al., 2007) Th2 polarization by DCs is mediated by

IL-4 (Le Gros et al., 1990; Swain et al., 1990), OX40L (Ito et al., 2005), IL-25 (Fort et al.,

2001) and IL-33 (Schmitz et al., 2005) The notch ligand families, Delta and Jagged,

induce Th1 and Th2 polarization respectively (Amsen et al., 2004)

T-bet is the central transcription factor involved the development of Th1 cells (Szabo et

al., 2000) T-bet potently activates the transcription of the IFN-γ gene, and its expression

is correlated with IFN-γ production by Th1 and NK cells IL-12 and IFN-γ triggers

STAT1 and STAT4 which eventually leads to the activation of T-bet (Afkarian et al.,

2002; Grogan et al., 2001; Lighvani et al., 2001) GATA-3 is the critical regulatory

transcription factor involved in Th2 differentiation which controls the transcription of

IL-4, IL-5, IL-6, IL-10 and IL-13 (Ouyang et al., 1998; Zhang et al., 1997; Zheng & Flavell,

1997) STAT6 is also a central mediator of IL-4 signaling and Th2 development that acts

upstream of GATA-3 (Hou et al., 1994; Quelle et al., 1995)

Polarization of the immune response by DCs

DCs are not only crucial for the initiation of T cell responses, they also determine the

subsequent character of the elicited T cell response This is exemplified by the three

signal concept (Kalinski et al., 1999) Signal one represents the antigen specific TCR

stimulation by MHC-peptide complexes formed through antigen processing and

presentation Signal two represents co-stimulatory signals, for instance CD28 stimulation

by CD80/86 that is essential for optimal T cell proliferation and activation Signal three

represents factors that determine the character of the T cell response i.e Th1, Th2, Th17

or Treg DCs can be activated by signals from various sources DCs express PRRs like

TLRs that recognize microbial PAMPs which activate immature DCs into professional

antigen presenting cells (Janeway & Medzhitov, 2002; Kapsenberg, 2003) Inflammatory

mediators, complement and other “danger” signals derived from tissue damage also

modulates DCs function (Gallucci & Matzinger, 2001; Lu et al., 2008) DC activity is

also influenced through direct and indirect interaction with other effector immune cells,

which results in the delivery of “feedback” signals that can influence the activation status

of the DC (Alpan et al., 2004; Kalinski & Moser, 2005; Munz et al., 2005) In order to

mount an immune response that is effective and appropriate, DCs must integrate these

signals during activation, which can act to synergize or regulate each other (Mosmann &

Livingstone, 2004) Fig 1.2 illustrates how the polarizing activity of DCs can be affected

by activating stimuli

Figure 1.2 DCs integrate signals from various sources and differentiate into Th1 or Th2

inducing DCs

“Danger signals”

from inflammation and necrotic tissue damage (eg complement, heat shock proteins)

Cell derived factors Interacting cells and microenviromental cues Contact dependent (eg CD40L)

or soluble factors (eg inflammatory cytokines)

Pathogenic signals

(eg TLR ligands)

Integration of signals

By DCs

Toll-like receptors

The innate immune system recognizes molecular signatures that are unique to

micro-organisms (Medzhitov & Janeway, 1997) These are conserved molecular patterns that

are called pathogen-associated molecular patterns (PAMPs) The receptors that recognize

PAMPs are pattern recognition receptors (PRR) The best studied of PRRs are receptors

of the toll-like receptor family (Medzhitov et al., 1997) TLRs are type I transmembrane

receptors characterized by an extracellular leucine-rich repeat (LRR) domain and an

intracellular Toll/IL-1 receptor (TIR) domain

To date, 12 TLRs have been identified in mouse and 10 TLRs in humans (Akira et al.,

2006) Collectively, TLRs respond to a broad range microbial products and endogenous

“danger” signals (Gallucci & Matzinger, 2001) TLR 2 associates with TLR1 which

recognizes bacterial triacylated lipopeptides, or with TLR6 that recognizes diacylated

lipopeptides, mycoplasma derived lipoproteins, proteoglycans and zymosan (Ozinsky et

al., 2000; Takeuchi et al., 2001) TLR3 recognizes dsRNA that produced by most viruses

during their replicative cycle (Alexopoulou et al., 2001) TLR4 recognizes LPS, the

bacterial cell wall component of gram negative bacteria (Poltorak et al., 1998; Qureshi et

al., 1999) TLR5 recognizes flagellin (Hayashi et al., 2001), TLR7 and 8 recognize

uridine or uridine/guanosine-rich ssRNA which can be found in ssRNA viruses (Diebold

et al., 2004; Heil et al., 2004) TLR9 recognizes unmethylated CpG motifs in bacterial

and viral DNA (Hemmi et al., 2000) Figure 1.3 illustrates the cellular location of the

different TLRs and their ligands

TLRs are mainly expressed by APCs, including DCs, macrophages, monocytes and B

cells They are also expressed by T cells and non-immune cells like epithelial cells and

fibroblasts (Takeda et al., 2003) TLRs are expressed extra or intracellularly TLR1, 2, 4,

5 and 6 are expressed on the cell surface, while TLR3, 7, 8 and 9 are found in

intracellular compartments Hence, their ligands, mainly nucleic acids, require

internalization before signaling is possible (Akira et al., 2006) Stimulation of TLRs

results in the recruitment of adaptor molecules such as myeloid differentiation factor 88

(MyD88) and TIRAP This then results the triggering of downstream signaling cascades

and the activation of NF-κβ, MAP kinases and IRFs, which results in the induction of

inflammatory genes and production of pro-inflammatory cyokines, chemokines and type

1 interferons (Eisenbarth et al., 2002; Kawai & Akira, 2007)

TLR stimulation and the development of Th1 and Th2 polarizing DCs

All TLRs share the common ability to induce inflammatory cytokines and up-regulate

co-stimulatory molecules, albeit to different extents Triggering of each TLR can have its

own specific effect, for example production of T cell polarizing factors like IL-12 There

is an explosion of interest on this issue of late, and the available literature is a daunting

mix of the effects of different TLR ligands in vitro, in vivo and different effects on

different cell types, including monocytes, macrophages and DCs Within DCs, effects of

TLR ligands on human MoDCs, mouse BMDCs, splenic DCs, sorted DC subsets have

been described These are summarized in several reviews and collectively, certain

reliable conclusions can be drawn (Agrawal et al., 2003; Kaisho & Akira, 2006;

Kapsenberg, 2003; Mazzoni & Segal, 2004; Pulendran, 2005)

Broadly TLR3, TLR7/8 and TLR9 stimulation results in strong Th1 responses, as the

stimulation of these TLRs results in the production of high levels of IL-12 and IFN-α

Ligands for TLR 2 and TLR5 have been reported to be weak IL-12p70, but strong IL-10

stimulators Hence, stimulation of TLR 2 and TLR5 seems to favor Th2 or Treg

responses The T helper response with LPS, the prototypical TLR4 ligand, is more

malleable, and is less potent than TLR3 and TLR9 in the induction of IL-12 For example

with LPS, low antigen doses induces Th2 responses, while high antigen doses induce Th1

responses (Boonstra et al., 2003; Eisenbarth et al., 2002; Langenkamp et al., 2000) IFN-γ

with LPS also strongly augments DCs to prime Th1 responses (de Jong et al., 2002)

Furthermore, LPS from different pathogens might induce different T helper responses

(Pulendran et al., 2001)

Mice lacking the TLR adapter MyD88 fail to mount effective Th1 responses (Fremond et

al., 2004; Kaisho et al., 2002) This had led to the conclusion that TLR signaling results

in Th1 responses by default However, it has been shown that transcription factors that

are triggered downstream of MyD88, for instance extracellular signal-regulated kinase

(ERK) and mitogen activated protein kinase (MAPK) suppresses IL-12p70 while

promoting IL-10 production, while c-Fos promotes IL-12p70 production (Agrawal et al.,

2003; Dillon et al., 2004) Hence, TLR activation can result in either Th1 or Th2

responses, depending on which transcription factors are eventually triggered

Recently, it has been shown that simultaneously activating different TLR ligands can

result in complementary, synergistic or antagonistic effects (Trinchieri & Sher, 2007)

Notably, synergy between TLR ligands is especially true for the case of bioactive

IL-12p70 While stimulation of a single TLR induced low levels of IL-12p40 and

insignificant levels of IL-12p70, simultaneous stimulation of TLR7/8 and TLR3 and 4

results the synergistic production of high levels of IL-12p70 (Gautier et al., 2005;

Napolitani et al., 2005) Importantly, a single pathogen possess several TLR ligands that

can activate multiple TLRs simultaneously (Trinchieri & Sher, 2007)

Propensity of different DC subsets to induce Th1 and Th2 responses

While all DC subsets can present antigen to and prime antigen specific T cells, the

outcome of this priming can vary considerably Human MoDCs were thought to induce

Th1 differentiation, while pDCs induced Th2 responses (Rissoan et al., 1999) Similarly

in mice, CD4-CD8α+ DCs have been shown to preferentially induce Th1 responses due to

their superior ability to produce IL-12p70 over the other DC subsets, whereas CD8α- DCs

tend to induce Th2 responses (Edwards et al., 2002; Hochrein et al., 2001; Iwasaki &

Kelsall, 2001; Maldonado-Lopez et al., 1999; Pulendran et al., 1999; Shortman & Liu,

2002) However, this functional dichotomy has been challenged by studies suggesting

that T cell polarizing activity of DCs is controlled by the nature of the activation signals,

for example different TLR ligands used for DC stimulation (Boonstra et al., 2003;

Edwards et al., 2002; Manickasingham et al., 2003; Pulendran et al., 2001) For instance,

pDCs induced strong Th1 responses when stimulated with influenza virus or CpG (Cella

et al., 2000; Krug et al., 2001) Similarly, human MoDCs are also capable of inducing

Th2 response with LPS (Jotwani et al., 2003)

Furthermore, different DC subsets express a different panel of TLRs For instance,

(Edwards et al., 2003) CD8α+ DCs also have the relatively low TLR5 mRNA and the

highest level of TLR3 (Reis e Sousa, 2004) Hence, different TLR ligands may

preferentially induce DCs to polarize different T cell subsets due to their preferential

ability to stimulate different DC subsets, for instance, CD8α+ DCs for Th1 and CD8α

-DCs for Th2 However, this simple conclusion is confounded by the fact that -DCs or its

subsets lacking the expression of a particular TLR can be activated by paracrine factors

produced by other DC subsets or cell type (Doxsee et al., 2003; Lebre et al., 2003) For

example, type I interferons can act in autocrine and paracrine fashion to stimulate DCs

(Hoshino et al., 2002; Le Bon et al., 2001; Montoya et al., 2002) In conclusion, although

different DC subsets have an innate propensity to produce T cell polarizing factors, the

DC1 DC2 dichotomy has yet to be shown to represent a real division The production of

these factors depends on whether and how these DC subsets are activated by the TLR

ligands

Modulation of DCs by interacting cells

The microenvironment, in particular the interaction of DCs with cells within their

microenvironment, is known to heavily influence the T helper inducing abilities of DCs

It seems that this can be a predominant, overriding force in particular in the presence of

weak TLR stimulation The lung microenvironment DCs are conditioned to induce

tolerance or Th2 responses Tolerance to harmless inhaled antigens was shown to be

mediated by lung resident pDCs, which was suggested to suppress the potential of

lung-cells has also been shown to be crucial in influencing the Th2 polarization activities of

DCs (Hammad & Lambrecht, 2008) Stimulation of epithelial cells via PRRs results in

the production of thymic stromal lymphopoietin (TSLP) that directly activates DCs to

prime CD4 T cells to differentiate into Th2 cells in a OX40L dependent mechanism (Ito

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

Of late, it has become increasing clear that other cells of the immune system can

reciprocally regulate the function of DCs In general, B cells interacting with DCs

produce IL-10 that induces Th2 promoting DCs (Fillatreau et al., 2002; Mizoguchi et al.,

2002; Moulin et al., 2000; Skok et al., 1999) DCs from B cell deficient mice produced

greater levels of IL-12 and exhibit enhanced Th1 responses (Moulin et al., 2000)

Conversely, cell mediated immunity can be enhanced by the binding of antigen IgG

complexes on FcγRI and FcγRIII on DCs that promotes cross-presentation of these

antigens (Regnault et al., 1999)

Mast cells are abundantly found in mucosal tissues and in close proximity to immature

DCs Mast cells secrete histamine in response to IgE-immune complexes that binds to

their Fcε receptors Histamine has a profound effect on DC by suppression of IL-12 and

promotion of IL-10 production by DCs, resulting in the induction of Th2 responses

(Caron et al., 2001; Mazzoni et al., 2001; van der Pouw Kraan et al., 1998) Similarly,

prostaglandin D2, another major product of activated mast cells, strongly suppresses the

ability of DCs to produce IL-12 (Faveeuw et al., 2003)

NK cells promote the activation of DCs that polarize Th1 responses Activated NK cells

produce TNF and IFN-γ that induces DC maturation and its Th1 polarization capability

(Degli-Esposti & Smyth, 2005; Gerosa et al., 2002; Mailliard et al., 2003; Mocikat et al.,

2003; Piccioli et al., 2002) Alternatively, NK cells can negatively regulate DCs by

killing them (Piccioli et al., 2002; Wilson et al., 1999)

It has been observed that mice lacking CD4 T cells fail to mount effective cytotoxic CD8

T cell responses It was discovered that that is was due to the ability of CD4 T cells to

“licence” DCs for stimulating the killing activity of CD8 T cells (Bennett et al., 1998;

Ridge et al., 1998; Schoenberger et al., 1998) The molecule involved in this licencing

process was CD40L, which triggers CD40 expression on DCs Similarly, mice lacking

CD40 or CD40L did not mount helper dependent killer CD8 T cell responses

Pre-stimulation of DCs with anti-CD40 in vitro, or the injection of anti-CD40 antibodies into

helper T cell deficient mice restored the ability of DCs to stimulate CD8 T cell killer

responses Stimulation of CD40 on DCs enhances the production of inflammatory

cytokines, especially IL-12 (Caux et al., 1994; Cella et al., 1996) Hence, CD40

triggering of DC by CD4 T cells results in the production of IL-12 that is important for

activating the killer activity of CD8 T cells

Modulation of DCs by CD8 T cells

CD8 T cells are known principally for their role in cell killing However, their ability to

promote Th1 responses has also been described In vivo, CD8 T cells have been shown to

induce protective Th1 responses against microbial infections For instance, during

Lesihmania major infection, it was observed that low dose infection resulted in Th2

responses CD8 T cells were required for the eventual down modulation of low dose

induced Th2 responses and resistance to L major (Uzonna et al., 2004) Similarly,

depletion of CD8 T cells abrogated the protective response induced by DNA vaccination

(Gurunathan et al., 2000; Salagianni et al., 2007) This was associated with a marked

decrease in Th1 cells, suggesting that CD8 T cells were important for the development of

Th1 cells During retroviral infection, CD8 T cells are also involved in the generation of

protective CD4 Th1 responses (Peterson et al., 2002) CD8 T cells also have the ability to

deviate the immune response away from allergic Th2 phenotypes Previous work from

our group had showed that the in vivo depletion of CD8 T cells in OVA/alum challenged

animals resulted in massive increases in IgE responses, whereas the adoptive transfer of

OVA specific CD8 T cells in CD8 T cell deficient mice results in a signifant reduction in

IgE (MacAry et al., 1998) It was later shown that CD8 T cell mediated IgE suppression

was lost in IL-12 and IL-18 deficient hosts, which could be restored by the transfer of

WT DCs (Salagianni et al., 2007; Thomas et al., 2002) These data suggests that CD8 T

cells induce DCs to produce IL-12 and IL-18 which suppresses the allergic IgE responses

CD8 T cells can have a direct impact on DCs during their interaction, inducing DCs to

mature (Ruedl et al., 1999), and enhance their Th1 polarizing capabilities by augmenting

their ability to produce IL-12p70 (Mailliard et al., 2002; Thomas et al., 2002) The ability

of CD8 T cells to enhance DC IL-12p70 production is attributed mainly to the production

of IFN-γ by CD8 T cells

Like NK cells, CD8 T cells can also negatively regulate DC activity by killing them

(Guarda et al., 2007) However, expression of protease inhibitors, like protease

inhibitor-9 and serpin serine protease inhbitor-6 prevents can protect DCs from killing by CD8 T

cells (Hirst et al., 2003; Medema et al., 2001) These proteases are up-regulated by DC

activating factors like LPS and TNF-α Interestingly, it has been shown that while

effector CD8 T cells kill DCs in a granzyme B and perforin dependent mechanism,

memory CD8 T cells protected DCs from killing (Nakamura et al., 2007; Watchmaker et

al., 2008) Memory CD8 T cells were found to release TNF-α early during interaction,

thereby inducing the expression of granzyme B inhibitor PI-9 and subsequently

protecting them from being killed by cytotoxic granules Therefore, it was shown in these

studies that memory but not effector CD8 T cells were able to prime DCs for IL-12 and

tumor protective immune responses (Nakamura et al., 2007; Watchmaker et al., 2008)

Aims of this study

It was previously shown using an in vivo OVA lgE model that that adoptively transferred

OVA specific CD8 T cells could suppress IgE responses (Thomas et al., 2002) This

ability was attributed to the ability of CD8 T cells to prime DCs for IL-12 production

CD8 T cells from IFN-γ-/- mice were able to suppress IgE responses as effectively as CD8

T cells from WT mice On the contrary, CD8 T cell derived IFN-γ was shown to be

absolutely necessary for induction of IL-12 by DCs (Mailliard et al., 2002) This suggests

that alternative mechanisms for CD8 T cell mediated IL-12p70 priming exists

Furthermore, DCs used for experiments thus far were that were generated from human

monocyte precursors or mouse bone marrow cells grown with GM-CSF and IL-4 These

cells only appear under inflammatory conditions and do not represent lymphoid resident

DCs (Shortman & Naik, 2007) Hence, it is unknown if CD8 T cells could possibly

modulate IL-12p70 production with splenic resident DCs, and if so, what other

mechanisms they could employ to mediate IL-12p70 production

Specific aims

1) To determine if CD8 T cells can induce DC IL-12p70 production and the

conditions that are necessary in vitro

2) To determine what CD8 T cell derived soluble/surface factors regulate DC