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The Maintenance of Immune Homeostasis and Quiescence by Negative Regulators of Immunity by Dylan James Johnson A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Department of Immunology University of Toronto © Copyright by Dylan Johnson 2015
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Page 1: The Maintenance of Immune Homeostasis and Quiescence by ...

The Maintenance of Immune Homeostasis and

Quiescence by Negative Regulators of Immunity

by

Dylan James Johnson

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

Department of Immunology University of Toronto

© Copyright by Dylan Johnson 2015

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The Maintenance of Immune Homeostasis by Negative

Regulators of Immunity

Dylan James Johnson

Doctor of Philosophy

Department of Immunology

University of Toronto

2015

Abstract

The immune system is tightly controlled by molecules that regulate the generation of

immunity. Additionally, these molecules may play important roles in regulating quiescent

immune cells. This thesis examines the role of several of these molecules in the biology of both

quiescent and activated immune cells.

The phosphatase Shp1 has been previously described as a negative regulator of T cell

receptor signaling. This thesis presents evidence that Shp1 does not directly regulate the

signaling from T cell receptor, but instead regulates T cell homeostasis and Th2 skewing. Shp1

controls both of these aspects of T cell biology through the regulation of IL-4 signals.

The Nfkb1 gene, which encodes for NFκB proteins p50 and p105, has been previously

demonstrated to maintain the quiescence of dendritic cells. The loss of Nfkb1 permits DCs to

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bypass the requirements for TLR-induced maturation for the induction of CD8+ T cell-mediated

immune pathology. This thesis delineates unique roles for p50 and p105 in the regulation of DC

biology. Furthermore, we demonstrate that the NFκB protein p50, but not p105, is essential for

maintaining the quiescence of immature dendritic cells.

The ubiquitin editing enzyme A20 negatively regulates NFκB activity downstream of

TLR signaling. This thesis demonstrates that A20, like Nfkb1, is also required to preserve the

dendritic cells in a functionally quiescent state. A20- and Nfkb1-deficient dendritic cells share a

core phenotype characterized by an ability to induce CD8+ T cell responses, low expression of

costimulatory molecules, increased basal TNF section, and greatly reduced expression of NFκB

proteins.

We herein present evidence demonstrating that the regulatory molecules Shp1, p50, and

A20 are required to preserve immune cells in their normal steady states. Immune cell quiescence

is therefore not a passive default state, but is instead actively regulated and maintained.

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Acknowledgments

It’s certainly been a journey - one that wouldn’t have been possible, or nearly as

enjoyable, without the countless contributions and endless support that I received from family,

friends, and colleagues.

To my supervisor, Pam: Thank you for your mentorship and guidance. You’ve cultivated

a lab environment that encourages curiosity, intellectual independence, collaboration and

comradery. While balancing your endless responsibilities, deadlines, and priorities, you still

found the time to provide support – and always with a smile. In particular, I will always be

grateful that you read this thesis with incredible speed to help me make my deadlines. Thank

you!

To the Ohashi Lab: Thank you for a truly great 6 years. Without a doubt we’ve created

some lifelong memories and friendships and I am going to greatly miss the daily antics and

happenings about the lab. My fellow grad students Charles, Heather, and Michael, I’m grateful

we had each other to experience the highs and endure the lows of graduate school. Without a

doubt, you’ll all rock the rest of your PhDs. Celine, thank you for always sharing DCs and for

being my partner in surviving the chaos of co-culture day. Ginny, you put up with me for longer

than I probably deserved and made immeasurable contributions to the lab. Special thanks to Cow

Bay co-founder Sarah and Secret Santa co-conspirator Carlos for all the laughs. Alisha, Tash,

Linh, Sara, Evan, Doug, Kiichi, Patty, James, Ramtin, Jessica, Mike – thank you all for your

contributions and positivity. It really wouldn’t have been the same without you.

Tim, I know you’ll be as happy as I am to be free of Sunday afternoon FACS dates. Your

limitless patience and support made this all possible. All the thanks, for now and for always.

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Table of Contents

Acknowledgments ......................................................................................................................................................... i

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

List of Figures ............................................................................................................................................................vii

Chapter I: Introduction .............................................................................................................................................. 1

Dendritic Cells ........................................................................................................................................................ 2

Diversity and Development ................................................................................................................................ 3

Maturation ........................................................................................................................................................... 5

Pattern-recognition receptors .............................................................................................................................. 9

The regulation of dendritic cell maturation by NFκB ....................................................................................... 15

Overview and Structure .................................................................................................................................... 15

Initiation through IKK activation ...................................................................................................................... 18

Sequestration and release by IκB proteins ........................................................................................................ 22

Modulation of transcription by NFκB dimers ................................................................................................... 24

Termination of NFκB signaling ........................................................................................................................ 27

Regulation through ubiquitination .................................................................................................................... 29

Regulation through A20 .................................................................................................................................... 32

T cell activation .................................................................................................................................................... 37

T cell Receptor Stimulation .............................................................................................................................. 37

Co-regulation .................................................................................................................................................... 41

The regulation of T cell activation by phosphorylation .................................................................................... 45

Kinases .............................................................................................................................................................. 45

Phosphatases ..................................................................................................................................................... 47

Sh2 domain-containing phosphatases ............................................................................................................... 50

Regulation of inflammation by Shp1 ................................................................................................................ 53

Regulation of T cells by Shp1 ........................................................................................................................... 55

Thesis outline and goals ....................................................................................................................................... 59

Chapter II: Materials and Methods ......................................................................................................................... 61

Mice .................................................................................................................................................................. 62

Western Blots and EMSA ................................................................................................................................. 62

Flow cytometry and Cell sorting ....................................................................................................................... 63

In vitro T cell assays ......................................................................................................................................... 64

BMDC Generation ............................................................................................................................................ 65

ELISAs and Bead Arrays .................................................................................................................................. 66

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Chapter III: Shp1 regulates T cell homeostasis by limiting IL-4 Signals ............................................................. 67

Introduction .......................................................................................................................................................... 68

Results ................................................................................................................................................................... 70

T cell-specific deletion of Shp1 ........................................................................................................................ 70

Thymocytes develop normally in the absence of Shp1 ..................................................................................... 72

Memory-phenotype T cells accumulate in Shp1 conditional knockout mice ................................................... 74

T cells respond normally to TCR stimulation in the absence of Shp1 .............................................................. 77

T cells skew to Th2 in the absence of Shp1 ...................................................................................................... 79

Memory-phenotype cells in Shp1 conditional knockout mice are dependent on IL-4 ...................................... 81

Discussion ................................................................................................................................................................... 85

Absence of Shp1 in T cells does not phenocopy the T cell phenotype of me mice .......................................... 85

Shp1 restricts the development of memory-phenotype T cells ......................................................................... 86

Shp1 negatively regulates Th2 skewing ............................................................................................................ 88

Concluding remarks .......................................................................................................................................... 90

Chapter IV: The NFκB subunit p50 limits the immunogenicity of dendritic cells ............................................. 92

Introduction .......................................................................................................................................................... 93

Results ................................................................................................................................................................... 96

Generation of DCs lacking NFκB p50 and p105 .............................................................................................. 96

Loss of p50 and p105 in DCs alters CD8+ T cell activation in vitro ................................................................ 99

Nfkb1-/-

DCs drive CD8+ T cell activation through antigen- and TNFα-dependent mechanisms .................. 101

Nfkb1-/-

DCs induce limited CD4+ T cell activation ...................................................................................... 104

Loss of p105 in unstimulated DCs does not impart the ability to induce diabetes .......................................... 106

Nfkb1 mixed bone marrow chimeras develop autoimmunity ......................................................................... 108

Discussion ............................................................................................................................................................ 113

NFκB p50 and p105 play distinct roles in DC biology ................................................................................... 113

DC expression of NFκB p50 is required to prevent CD8+ T cell-mediated pathology .................................. 115

Nfkb1 chimerism disrupts T cell homeostasis and promotes inflammation ................................................... 117

Concluding Remarks ....................................................................................................................................... 118

Chapter V: A20 and the molecular regulation of NFκB ...................................................................................... 120

Introduction ........................................................................................................................................................ 121

Results ................................................................................................................................................................. 124

Generation of A20 deficient dendritic cells .................................................................................................... 124

A20 is required for conventional dendritic cell maturation ............................................................................ 124

Loss of A20 in DCs has minor impact on in vitro T cell activation ................................................................ 127

A20 maintains DC quiescence ........................................................................................................................ 129

The expression of NFκB proteins is ablated in Nfkb1- and A20-deficient DCs ............................................. 132

Inhibition of the proteasome partially restores NFκB protein expression in Nfkb1-deficient DCs ................ 137

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Discussion ............................................................................................................................................................ 139

A20 is required to preserve DC quiescence .................................................................................................... 139

A20 plays dual roles during DC maturation ................................................................................................... 142

A20 and p50 maintain expression of NFκB .................................................................................................... 143

Concluding Remarks ....................................................................................................................................... 146

Chapter VI: Discussion ........................................................................................................................................... 147

The regulation of homeostatic signals ............................................................................................................ 148

Active regulation of immune cell quiescence ................................................................................................. 150

Destabilization of the immature DC state ....................................................................................................... 151

The ablation of NF𝜅B expression in destabilized DCs ................................................................................... 155

Co-stimulation of T cell responses.................................................................................................................. 156

Concluding Remarks ....................................................................................................................................... 158

References ................................................................................................................................................................ 159

Copyright Information ............................................................................................................................................ 217

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List of Figures

Figure I-1. Toll-like receptor engagement stimulates a signaling cascade

leading to the activation of transcription factors .................................................................................................. 11

Figure I-2. Members of the NFκB transcription factor family ............................................................................ 17

Figure I-3. Activation of canonical NFκB signaling.............................................................................................. 20

Figure I-4. T cell receptor engagement initiates a cascade of signaling events .................................................. 39

Figure III-1. T cell specific deletion of Shp1.......................................................................................................... 71

Figure III-2. Thymocytes develop normally in the absence of Shp1 ................................................................... 73

Figure III-3. Shp1 restricts the development of memory-phenotype T cells ...................................................... 86

Figure III-4. Shp1-deficient T cells exhibit normal responses to TCR stimulation ........................................... 78

Figure III-5. T cells skew to Th2 in the absence of Shp1 ...................................................................................... 80

Figure III-6. IL-4 is required for the accumulation of CD44hi

T cells in

Shp1 conditional knockout mice ............................................................................................................................. 83

Figure IV-1. Generation of DCs lacking NFκB p50 and p105 ............................................................................. 97

Figure IV-2. CD8+ T cell activation is altered by loss of NFκB1 proteins ......................................................... 100

Figure IV-3. Antigen- and TNFα-dependent mechanisms drive

T cell activation by NFκB1-deficient DCs ............................................................................................................ 103

Figure IV-4. Nfkb1-deficient DCs fail to induce Smarta T cell activation. ...................................................... 105

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Figure IV-5. Loss of p50 expression is required for unstimulated DCs to

induce diabetes in RIP-gp mice ............................................................................................................................. 107

Figure IV-6. Nfkb1 bone marrow chimeras have altered T cell homeostasis .................................................... 109

Figure IV-7. Wild type T cells are activated in Nfkb1 chimeric mice ................................................................ 112

Figure V-1. Generation of A20-deficient DCs ...................................................................................................... 126

Figure V-2. Dendritic cell A20 has minimal impact on in vitro P14 activation ................................................. 128

Figure V-3. Unstimulated A20-deficient DCs induce diabetes in RIP-gp mice ................................................. 131

Figure V-4. A20 and Nfkb1-deficient DCs have reduced expression of NFκB proteins ................................... 133

Figure V-5. Nfkb1-deficient DCs do not have elevated expression of

TNFα transcription factors .................................................................................................................................... 136

Figure V-6. NFκB proteins are degraded in Nfkb1-deficient DCs ...................................................................... 138

Figure VI-1. A destabilized DC phenotype induces T cell immunity ................................................................. 154

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Chapter I

Introduction

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The mammalian immune system is an intricate web composed of a myriad of cells, molecules,

and their interactions. These elements endow the immune system with the ability to react, or not,

to ourselves, pathogens, and environmental antigens. In the absence of an immunological insult,

cells of the immune system are in a state of quiescence. However, upon the activation of the

immune system, leukocytes undergo rapid cellular proliferation and differentiation. This cellular

dynamism is unparalleled by any other body-system and underpins the generation of immunity.

The maintenance of health is contingent upon appropriate regulation of the various arms

of immunity. A failure of a regulatory mechanism to appropriately control the generation or

differentiation of an immune response can have severe consequences including the generation of

autoimmunity, inflammation, or immunodeficiency. The identification and characterization of

the molecules responsible for controlling these processes is therefore of fundamental importance.

We will herein focus on the control of T cell biology, through both intrinsic regulatory elements

and the activation of dendritic cells.

Dendritic Cells

Dendritic cells are a core component of the mammalian immune system, coordinating the

activation of adaptive immunity. Since their discovery by Steinman and Cohn [1, 2], the ability

of dendritic cells to control both the maintenance of immune tolerance and the induction of

immunity has been firmly established. Dendritic cells are innate cells that act as immunological

scouts, whereby they acquire peripheral antigens and present them to lymphocytes for

recognition. Furthermore, they integrate signs of infection and injury to alternatively promote

immune tolerance or the induction of potent adaptive immune responses upon their presentation

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of antigens to adaptive immune cells. As such, dendritic cells are the true conduit between the

innate and adaptive branches of immunity. The regulation of dendritic cell biology is therefore

paramount to the maintenance of health both during homeostasis and immunological insult.

Diversity and Development

Dendritic cells are a heterogeneous group of hematopoietic cells characterized by a diversity of

functions and localizations[3]. Dendritic cells are broadly categorized as either plasmacytoid

(pDC) or conventional (cDC). pDCs, found primarily in the blood and lymphoid organs, are

characterized by their relatively low expression of Major histocompatibility complex class II

(MHCII) molecules and costimulatory molecules. Upon recognition of nucleic acids, pDCs

possess the ability to produce large quantities of type I interferons (IFNs). Accordingly, pDCs

have been found to play a major role during viral infections [4]. cDCs can be further

subcategorized into various groups based on their ontogeny, tissue localization, and functional

specialization. However, all cDCs share a core set of features including expression of CD11c,

their capacity to acquire and process antigen for presentation, a tendency to migrate to T cell

zones of lymphoid organs, and superior ability to prime the activation of naïve T cells [3]. A

focus of this thesis is the biology of cDCs, which from here on will be referred to simply as DCs.

DCs are further characterized by their localization. One group of DCs, termed migratory

DCs, are most often found within non-lymphoid tissues where they constantly acquire and

process antigen [3]. Although these tissue-resident DCs do occasionally migrate to lymphoid

tissues during steady-state, their migration to local draining lymph nodes is greatly increased

following their activation [5]. Migratory DCs are further subdivided by their expression of either

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CD103 or CD11b. Lymphoid tissue-resident DCs are the other major population of conventional

DCs during steady-state [3]. These DCs are found within the spleen, lymph nodes and mucosa-

associated lymphoid tissues. Lymphoid tissue-resident DCs may also be divided by their

exclusive expression of one of two markers, in this case, either CD8 or CD11b. In spite of their

distinct surface phenotypes, migratory CD103+ and lymphoid resident CD8+ DCs appear to

have shared functional properties. Both populations appear to be particularly equipped for

efficient cross-presentation of antigens, activation of naïve CD8+ T cells, and the induction of

Th1 immunity [6]. The functional specialization of CD11b+ DCs is less clear although they are

known to be potent inducers of CD4+ T cell activation and may play important roles during the

induction of Th2 and Th17 immune responses [7].

Most DC populations are short-lived and therefore require constant replenishment

through the differentiation of bone marrow cells [3]. This developmental process is characterized

by a progressive differentiation into DCs and a concomitant loss of potential for alternative

lineages. The majority of DCs are likely derived from the common myeloid progenitor (CMP).

CMPs subsequently differentiate into macrophage-DC precursors (MDPs) which maintain the

potential for both monocyte/macrophage and DC differentiation. The MDP may give rise to a

common DC progenitor (CDP), a precursor to both pDCs and the various lineages of cDCs. The

progressive development of DCs is controlled by various cytokines. Fms-like tyrosine kinase 3

ligand (Flt3L) has been identified as a key cytokine promoting the differentiation of the CMP

into the CDP and subsequent DC lineages [3]. Accordingly, DC development is impaired in its

absence [8, 9]. Furthermore, Flt3L has been demonstrated to be important for the maintenance of

DC populations during homeostasis [9]. Additionally, granulocyte-macrophage colony-

stimulating factor (GM-CSF) has a demonstrated role in DC development. While GM-CSF-

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deficiency has minor impact on the development of lymphoid tissue resident DCs, migratory

DCs populations of the lung, skin, and intestine are considerably reduced [10]. GM-CSF has

been suggested to promote the ability of DCs to cross-present antigens [11]. Furthermore, GM-

CSF can promote monocyte differentiation into a CD11b+ DC population known as

inflammatory DCs [12, 13].

The identification of Flt3L and GM-CSF as key cytokines driving DC differentiation has

led to the development of in vitro DC generation protocols [14-16]. These methods allow for the

generation of large numbers of DCs, greatly facilitating basic DC research. Additionally, these

protocols can be used to generate clinically relevant numbers of DCs for therapeutic applications

[17]. Culture of mouse bone marrow cells with GM-CSF leads to the generation of DCs with a

phenotype reminiscent of CD11b+ lymphoid tissue-resident DCs. However, microarray analysis

has suggested that these in vitro generated DCs may be most similar to inflammatory monocyte-

derived DCs [18]. Alternatively, the generation of DCs with Flt3L leads to a mixed phenotype of

CD8+ and CD8- cells as well as a small proportion of cells with a pDC phenotype [16].

Maturation

DCs can carry out both tolerogenic and immunogenic functions. Based on observations that DC

populations can either be proficient at antigen uptake and presentation or potent inducers of

mixed leukocyte reactions, Steinman was the first to suggest that DCs can exist in two distinct

functional states [19]. This concept has since been elaborated into a central paradigm of DC

biology. By default, DCs exist in a tolerogenic state and are labeled “immature”, “resting”,

“quiescent”, or “steady state”. Through the integration of environmental cues, DCs become

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“mature” or “activated” and acquire the ability to induce immune responses. Understanding the

triggers and effectors of both tolerogenic and immunogenic DCs is therefore at the core of

understanding DC biology.

In the absence of infection, DCs exist primarily in an immature state. Experiments

demonstrated that in the absence of maturation stimuli, APCs fail to induce T cell immunity [20].

Furthermore, experiments wherein targeted delivery of antigens to endogenous DCs, through the

use of antibodies against DC surface molecules, demonstrated that immature DCs induce

antigen-specific T cell tolerance [21, 22]. Subsequent work demonstrated that immature DC-

induced tolerance is robust. Mice were engineered to express lymphocytic choriomeningitis virus

(LCMV) antigens in their DCs. Expression of LCMV antigens by DCs resulted in profound T

cell tolerance which could not be breached through infection with LCMV [23]. Immature DCs

maintain tolerance by mediating the induction of anergy, a state of unresponsiveness [24], the

deletion of T cells [25], the conversion of naïve CD4+ cells into regulatory T cells [26, 27], as

well as the maintenance of regulatory T cell populations [28, 29].

While there was an appreciation that the transition of a DC from an immature to mature

state was induced, the exact nature of the signal required for this process was not immediately

clear. The identification of mammalian Toll-like receptors (TLRs) provided the first clear

explanation of how the DC maturation process was induced. TLRs were found to recognize

microbial products such as lipopolysaccharide (LPS) and consequently induce innate immune

cells to express pro-inflammatory cytokines such as interleukin-6 (IL-6) as well as T cell

costimulatory molecules such as CD80 [30, 31]. The ability of TLR ligands was then confirmed

to induce in vivo maturation of DCs, providing the first illustration of the mechanism responsible

for the induction of DC immunogenicity [32, 33]. Subsequent work has demonstrated that TLRs

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are part of a larger class of molecules known as pattern recognition receptors (PRRs). The

various PRRs, to be covered in more detail below, detect a variety of molecules associated with

infection or cell death. Their ligation promotes activation of signaling cascades such as NFκB

which are thought to drive the maturation process. The DC maturation process is associated with

various morphological, phenotypical, and functional changes. Initiation of DC maturation results

in a transient increase in antigen uptake [34]. During this period, DC motility is also reduced

[35, 36]. Together, these two phenomena have been suggested to facilitate the uptake of antigen

from the site of PRR stimulation [37]. These changes, however, are temporary; within several

hours DCs reduce their uptake of exogenous antigen and concomitantly alter their regulation of

MHC molecules. In immature DCs, peptide-MHC complexes are rapidly turned over by non-

specific micropinocytosis [38]. Following their activation, however, these complexes are

stabilized increasing the total surface expression of MHC as well as maintaining presentation of

antigens acquired at the time of activation [39]. DC activation also prompts changes in the

expression of chemokine receptors and adhesions molecules that leads to their migration to

secondary lymphoid organs. Activated migratory DCs are believed to enter lymph nodes

primarily through the afferent lymphatics. This migration has been suggested to require

inflammatory cytokine signaling as both TNFα and IL-1β have been shown to be necessary [40,

41]. DCs undergoing maturation upregulate CCR7, facilitating migration towards lymphoid

organs by means of the chemokines CCL19 and CCL21 [42]. Furthermore, upregulation of the

chemokine receptor CXCR4 also contributes to DC homing to secondary lymphatics [43]. The

entire process of transient immobility followed by secondary lymphoid organ homing has been

suggested to take from 12 to 18 hours for migratory DCs [44].

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DC maturation also leads to the production of various soluble mediators that promote

inflammation and lymphocyte activation. Major contributors include the cytokines interleukins-6

(IL-6), -12 (IL-12), tumour necrosis factor (TNFα), and interferon α (IFN α). Different PRR

ligands appear to have distinct effects on the profile of cytokines produced by activated DCs. For

example, while peptidoglycan can induce robust production of TNFα, IL-6 and IL-1β, it is a very

poor stimulator of IL-12 production [45]. By contrast, CpG stimulation results in ample IL-12

production and little to no production of IL-1β [45]. The cytokine profile produced by DCs has a

direct impact in shaping the differentiation of an effector T cell response [45-47]. Therefore,

deciphering how distinct PRR ligands differentially regulate DC gene expression is key to

understanding the initiation of different immune response profiles.

Activation of DCs also promotes upregulation of cell surface molecules which are

believed to modulate their ability to stimulate T cells. These include members of the Ig-

superfamily, such as CD80 (B7.1), CD86 (B7.2), and inducible costimulatory molecule ligand

(ICOSL), as well as the tumour necrosis factor super family (TNFSF), such as 4-1BBL, OX40L,

CD40, CD70. Many of these, including CD80 and CD86, are thought to promote T cell

activation by providing co-stimulatory signals that enhance activation. Alternatively, some

molecules may direct signaling into the DC during DC:T cell interactions. For example, DC-

expressed CD40 is believed to interact with CD40L on T cells. This interaction leads to

activation of non-canonical NFkB signaling in the DC, promoting its ability to induce cytotoxic

T cell responses [48].

Several studies have reported DC populations that are phenotypically mature, but do not

induce immunity [49-51]. One common property of these mature DCs is that they do not produce

inflammatory cytokines, suggesting that upregulation of costimulatory molecules in the absence

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of inflammatory cytokines is insufficient to promote T cell activation. Furthermore, in some

settings, phenotypically mature DCs have been suggested to promote immune tolerance [52].

One study demonstrated that TNFα-treated DCs, which upregulated expression of CD80 and

CD86, were able to induce tolerance in the experimental autoimmune encephalomyelitis model

of multiple sclerosis. This was a feat that both immature and TLR-matured DCs could not

perform. Therefore, while these molecules are upregulated on activated DCs and they can

promote T cell activation, their expression may not always coincide with the generation of T cell

responses.

Pattern-recognition receptors

Triggering the innate arm of the immune system is intimately tied to the function of PRRs which

sense the byproducts of infection, cellular stress, tissue injury, or necrosis. PRRs are a

heterogeneous group of receptors which have distinct agonists, expression patterns, cellular

localizations, and signaling mechanisms. DCs have the ability to link the activation of PRRs with

the induction of adaptive immunity. Many PRRs are expressed by DCs and their engagement

induces dramatic alteration to DC function. Therefore, by understanding the signaling and

regulatory pathways activated by PRRs we can begin to appreciate the molecules which directly

control the transition of a DC from tolerogenic to immunogenic.

The most thoroughly studied PRRs in DC biology are the TLRs, a family of

transmembrane proteins that detect diverse ligands of bacterial, viral, fungal, and parasitic

origins. These pathogen-associated molecular patterns (PAMPs) are recognized by series of

leucine-rich repeats found within the ectodomain of all TLRs. PAMPs recognized by TLRs

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include lipoproteins (TLR1, TLR2, TLR6), RNA (TLR3, TLR7, TLR8), LPS (TLR4), flagellin

(TLR5), DNA (TLR9) [53]. Additionally, some TLRs have been implicated in detection of

damage-associated molecular patterns (DAMPs) such as high mobility group protein box 1

(HMGB1) (TLR4) and endogenous nucleic acids. Through TLR ligation, a signaling cascade

emerges which ultimately leads to the activation of pro-inflammatory transcription factors. All

TLRs contain a Toll-IL-1 receptor (TIR) domain in their cytoplasmic regions. These TIR

domains, contained within the cytoplasmic region of TLRs, are responsible for the recruitment of

TIR-containing adaptor proteins which are required for the propagation of signaling. TLRs can

be broadly classified by their dependence on one of two adaptor proteins. The majority of TLRs,

all those except for TLR3, can utilize myeloid differentiation response gene 88 protein (MyD88).

Alternatively, TLR3 and TLR4 may employ the TIR-domain-containing adaptor inducing IFN-β

(TRIF) as a signaling adaptor.

MyD88-dependent signaling is initiated by TLRs expressed both at the cell surface

(TLR1, TLR2, TLR4, TLR5, TLR6) and those contained within intracellular vesicles (TLR7,

TLR8, TLR9) [53] (Figure I-1). In some cases, MyD88 is directly recruited to the TIR domain

of the TLR. Alternatively, as is the case for TLRs 1, 2, 4, and 6, TIR-domain-containing adaptor

protein (TIRAP) may act as an adaptor, facilitating the recruitment of MyD88 to the receptors

intracellular domain. MyD88 contains a death domain (DD), which through homotypic

interactions with other DD-containing proteins, recruits IL-1 receptor-associated kinases

(IRAKs) to the TLR signaling complex. Therefore, MyD88 functions through linking innate

sensing of PAMPs to enzymatic activation.

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Figure I-1. Toll-like receptor engagement stimulates a signaling cascade leading to the

activation of transcription factors. The activation of TLRs promotes intracellular signaling

through recruitment of one of two signaling adaptors. Some TLRs, such as TLR7, TLR9, and

extracellular TLR4 recruit the adaptor protein MyD88. This leads to the activation complexes

containing IRAK proteins as well as TRAF E3 ubiquitin ligases. TRAF6-containing complexes

promote the activation of the TAK1 kinase complex which leads activation of MAPK signaling

as well as the IKK complex. The activation of the IKK complex initiates canonical NFκB

signaling. MyD88-dependent signaling may additionally lead to IKK1-mediated activation of

IRF7. Some endosomal TLRs such as TLR4 and TLR3 signal through recruitment of the adaptor

protein TRIF. TLR engagement subsequently leads to a TRAF3-mediated activation of IRF3 as

well as activation of the TAK1 complex and canonical NFκB signaling through RIP1.

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The first kinase activated downstream of MyD88-dependent signaling is IRAK4 which

subsequently leads to the activation of IRAK1 and IRAK2 [54]. The activation of IRAK proteins

results in recruitment of the E3 ubiquitin ligase, tumour necrosis factor receptor-associated factor

6 (TRAF6) [55]. TRAF6 catalyzes the formation of K63-linked polyubiquitin chains on itself as

well as IRAK proteins. This promotes the formation of a kinase complex composed of

transforming growth factor β-activated kinase 1 (TAK1) and the proteins TAK1-binding

proteins-2 and -3 (TAB2, TAB3) which directly bind to the K63-linked polyubiquitin chains.

This TAK1 complex may then go on to phosphorylate various targets to induce downstream

signaling. One target of TAK1 is the inhibitor of κB kinase (IKK) complex. Its phosphorylation

is required for the activation of NFκB pathway, which we will explore in more detail in the

ensuing section. TAK1 also been suggested to contribute to the activation of mitogen-activated

protein kinase (MAPK) cascades which drives the activation of the pro-inflammatory

transcription factor activator protein 1 (AP-1) [56].

TLRs 3 and 4 can both signal through MyD88-independent pathways by virtue of their

ability to recruit the adaptor protein TRIF. While TLR3 binds to TRIF directly through its TIR

domain, TLR4 uses the adaptor TRIF-related adaptor molecule (TRAM) to recruit TRIF. Upon

TLR ligation, TRIF may activate TRAF6, leading to the activation of the TAK1 complex as in

MyD88-dependent signaling. However, TRIF also leads to the recruitment of receptor-

interaction protein 1 (RIP1) which also contributes to the activation of the TAK1 complex.

Indeed, RIP1 mediated activation of TAK1 may be dominant in TRIF-dependent signaling as the

loss of RIP1, but not TRAF6, dramatically impaired TLR3 induced NFκB activation [57, 58].

TRIF also recruits the E3 ligase TRAF3 following TLR3 or TLR4 ligation [53]. TRAF3

can then promote the expression of type 1 interferons through the activation of tank-binding

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kinase 1 (TBK1) and IKKε. These kinases phosphorylate interferon regulatory factors-3 and -7

(IRF3, IRF7) which may then translocate into the nucleus and drive transcription of type 1

interferons. Although TRIF-independent, TLRs 7, 8, and 9 may also induce the activation of

TRAF3 and subsequently, production of interferons.

Several additional families of PRR contribute to the innate sensing of PAMPs and

DAMPs by DCs. The C-type lectin receptors (CLRs) form a large family of cell surface

molecules primarily involved in the recognition of carbohydrate moieties [59]. CLRs can induce

signaling through several distinct pathways, with the recruitment of the Syk kinase to ITAMs

being the most common. The consequences of CLR signaling are diverse, and depending on

which CLR is triggered may include phagocytic, inflammatory, and anti-inflammatory functions

[59]. One important role of CLRs is mediating the uptake and processing of glycosylated

antigens. CLR ligation, however, does not necessarily induce DC maturation. Consequently,

CLR-mediated antigen capture and presentation in the absence of TLR signaling may result in T

cell tolerance [21].

Cytoplasmic PRRs also contribute to the innate detection of PAMPs. The RIG1-like

receptors (RLRs), including retinoic acid-inducible gene 1 (RIG1) and melanoma differentiation-

associated gene 5 (MDA5), detect cytoplasmic RNA leading to the expression of type I

interferons and activation of NFκB by means of the adaptor protein mitochondrial antiviral

signaling proteins (MAVS) [60]. Interferon production in cDCs in response to viral RNA are

impaired in the absence of RIG1, suggesting that this pathway is crucial for antiviral responses in

cDCs [61]. By contrast, RNA-sensing TLRs were found to be more important in pDCs.

Another family of PRRs, the NOD-like receptors (NLRs), detects cytoplasmic

peptidoglycan from bacteria as well as a variety of DAMPs [62]. The prototypic members,

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nucleotide-binding oligomerization domain-1 and -2 (NOD1, NOD2) activate NFκB through

recruitment of the essential adaptor RIP2. However, other members of the NLR family, such as

NLRP2/4 have been suggested to inhibit NFκB activation by disrupting ubiquitination of TRAF6

[63, 64]. NLRs also mediate the processing of inflammatory cytokines through induction of

inflammasome assembly. Inflammasome-activating NLRs respond to diverse PAMPs and

DAMPs such as extracellular ATP, alum, uric acid crystals, and flagellin. Their activation results

in assembly of the inflammasome, a multiproten complex characterized by catalytically active

cysteine-aspartic protease-1 (Caspase1), which cleaves precursor proteins to form mature IL-1β

and IL-18. In summary, PRRs allow for the detection of diverse PAMPs and DAMPs to both

drive and modulate the activation of DCs. A majority of these PRRs promote NFκB, underlining

its fundamental role in DCs maturation.

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The regulation of dendritic cell maturation by NFκB

The NFκB signaling axis is a ubiquitous feature of all cells types. NFκB transcription factors are

the most thoroughly studied mediators of stimulus-responsive gene regulation. The induction of

NFκB activation is driven by diverse stimuli including PAMPs, DAMPs, pro-inflammatory

cytokines and ligands, B and T cell antigen receptors, metabolic and genotoxic stress, and

developmental cues. The impact of NFκB activation is equally broad; responses driven by NFκB

include cell survival, differentiation, proliferation, and cytokine production. Their impact is

perhaps most widely appreciated in immune cell signaling where they are known as master

regulators of inflammatory signaling. Furthermore, NFκB signaling is triggered by all known

stimuli of DC maturation, highlighting its fundamental importance in DC biology.

Overview and Structure

NFκB transcription factors are comprised of 5 proteins, namely p50, p52, p65, RelB, and cRel

[65, 66] (Figure I-2). All 5 NFκB members contain an N-terminal Rel homology domain

(RHD). These domains mediate the homo-and heterodimerization of NFκB proteins, forming

NFκB transcription factors. The activation of these NFκB transcription factors is tightly

regulated through several means. The chief mechanism is spatial restriction; NFκB dimers are

constitutively associated with IκB proteins within the cytoplasm. Signaling from NFκB-

activating stimuli converge upon the activation of IKK complexes. These activated kinase then

phosphorylate IκB proteins, leading to their consequential ubiquitination and proteasome-

mediated degradation. As a result, NFκB dimers are free to translocate into the nucleus where

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they bind to κB consensus sites within DNA through their RHDs and thereby direct the

transcription of target genes.

Based on dependence on the IKK regulatory subunit NEMO, NFκB signaling has been

conceptually divided into canonical (NEMO-dependent) and non-canonical (NEMO-

independent) signaling. Canonical NFκB is largely thought to be the result of signaling through a

wide range of inflammatory cytokine receptors, antigen receptors, and PRRs and prototypically

involves the activation of p50-, and p65-containing dimers. Non-canonical signaling primarily

induces activation of p52:RelB dimers and has been shown to mediate signaling downstream of

receptors such as CD40 and lymphotoxin β receptor (LTβR) where it plays a crucial role during

lymphoid organogenesis.

NFκB subunits can be divided based on the presence of transactivation domains. The

NFκB proteins produced by Rel (cRel), Rela (p65), and Relb (relB) all contain C-terminal

transactivation domains (TADs). Upon DNA binding, these TADs promote the transcription of

target genes. RelB additionally contains a leucine zipper (LZ) at its N-terminus. In addition to its

TAD, RelB requires this LZ for its full activation [67].

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Figure I-2. Members of the NFκB transcription factor family. Five mammalian genes

encode for NFκB proteins. All NFκB gene products contain a RHD which mediates the

dimerization of NFκB proteins as well as interaction with DNA. Three of the NFκB genes, Rel,

Rela, and Relb are translated directly into the NFκB subunits cRel, p65, RelB. These three

subunits contain TAD which allows them to initiate transcription. Nfkb1 and Nfkb2 are

transcribed into the precursor proteins p105 and p100. By virtue of their ARDs, these proteins

may act as IκB proteins, binding to other NFκB subunits in the cytoplasm. Proteolytic cleavage

of p105 and p100 may produce the NFκB subunits p50 and p52 which can homo- or

heterodimerize with other subunits to form NFκB transcription factors.

The two additional genes, Nfkb1 and Nfkb2, have several distinct properties which

distinguish them from the other NFκB genes [65, 66]. The gene products of both Nfkb1 and

Nfkb2 are precursor proteins, namely p105 and p100. These precursors may be processed into the

NFκB subunits p50 (from p105) and p52 (from p100). These subunits lack the transactivation

domains of other NFκB proteins. Consequently, NFκB dimers uniquely containing exclusively

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p50 and/or p52 cannot directly activate transcription. However, through heterodimerization with

TAD-containing subunits, p50 and p52 may contribute to transcriptional activation. While p105

and p100 contain the characteristic RHD of other NFκB proteins, they also contain ankyrin

repeat domains (ARDs) which are characteristic of IκB proteins. Indeed, both p105 and p100

display IκB-like properties, potentially binding to NFκB proteins in the cytoplasm, thereby

controlling their nuclear translocation.

Distinct mechanisms regulate the processing of p105 and p100 [65, 66]. Production of

p50 and p52 from their precursor proteins is a constitutive process mediated by ubiquitin-

dependent proteosomal degradation. A glycine rich region (GRR) within p105 and p100 prevents

their complete degradation, resulting instead in the release of the p50 and p52 subunits [68].

While the majority of p105 is constitutively processed into p50, the major product of Nfkb2

during steady-state appears to be p100. Alternatively, p105 may be targeted for degradation

during NFκB activation in a manner analogous to conventional IκB proteins. Unlike the

constitutive mechanism that results in p50 production, this process results in total degradation of

p105. The processing of p100 following stimulation is dependent on NFκB-inducing kinase

(NIK) and is a key feature of non-canonical NFκB signaling. NIK-induced degradation of p100

is incomplete, releasing p52-containing NFκB dimers to translocate to the nucleus and direct

gene expression.

Initiation through IKK activation

Signaling cascades triggered by many distinct cell-surface and intracellular receptors induce

activation of NFκB. Regardless of the origin of the signal, these pathways converge on the

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activation of IκB kinase (IKK) complexes which are responsible for inducing the

phosphorylation and degradation of IκB proteins [69]. The IKK complex is variably composed of

up to three distinct proteins. IKK1 (IKKα) and IKK2 (IKKβ) are two structurally homologous

proteins which confer catalytic function to the IKK complex. The third protein is NEMO, a

regulatory subunit lacking in any catalytic function. Canonical NFκB signaling activates a

tetrameric IKK complex invariably containing a NEMO dimer in addition to a homo- or

heterodimer of IKK1 and/or IKK2. By contrast, non-canonical signaling proceeds through

activation of IKK1 homodimers without the regulatory NEMO subunit. We will herein focus on

activation of the canonical IKK complex.

Dependence on NEMO is a defining feature of canonical NFκB signaling.

NEMO-deficiency leads to embryonic lethality due to an inability of fetal liver cells to activate

NFκB in response to TNFα [70, 71]. Furthermore, cells lacking NEMO are unable to activate

NFκB in response to inflammatory cytokines or TLR ligands [71, 72]. The capacity of NEMO to

support NFκB activity hinges upon its ability to bind to K63-linked polyubiquitin chains;

mutation of residues required for this binding inhibits NFκB activation [73]. Therefore, NEMO

may promote the activation of NFκB by bringing the IKK complex in association with

polyubiquitinated signaling intermediates. NEMO may also act to repress IKK activation in

certain settings. It has been reported that compromising the ability of IKK2 to bind to NEMO

results in enhanced catalytic activity and NFκB activation [74]. Therefore, NEMO provides both

positive and negative regulation to the IKK complex, providing control over NFκB activity.

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Figure I-3. Activation of canonical NFκB signaling. Signaling downstream of many immune

cell receptors including TLRs, NLRs, TCR, BCR, and TNFSFRs converge on the activation of

the IKK complex. This complex contains the regulatory subunit NEMO as well as the kinases

IKK1 and IKK2. The activated IKK complex leads to the phosphorylation of IκB proteins. This

event facilitates the recruitment of the SCF-βTrCP ubiquitin ligase complex which catalyzes the

formation of a K48-linked polyubiquitin tail on IκB. Proteasome mediated degradation of IκB

releases NFκB dimers allowing for their translocation into the nucleus. While unstimulated cells

may contain nuclear p50:p50 homodimers which may inhibit transcription, canonical signaling

leads to TAD-containing NFκB dimers, such as p65:p50 binding to DNA. These NFκB dimers

drive the transcription of various genes regulating cellular proliferation, differentiation, survival,

and inflammation. Additionally, NFκB activity induces the expression of several genes that form

a negative feedback loop in order to terminate signaling. Among these genes are IκBα, which

antagonizes the activities of NFκB dimers, and A20, which inhibits NFκB activity through the

regulation of ubiquitination of NFκB signaling intermediates.

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Several lines of evidence indicate that IKK2 is more important than IKK1 for canonical

signaling. IKK2 knockout mice have a more severe phenotype than IKK1 knockout mice, which

has been linked to their inability to respond to TNFα signals [75, 76]. Furthermore, IKK2 can

support NEMO-dependent NFκB activation in the absence of IKK1 [77]. However, some

inducers of canonical NFκB activation have been reported to be IKK1-dependent [78] [79],

suggesting that the differential requirements for IKK1 and IKK2 may be cell- or stimulus-

specific. In DCs, the loss of IKK1 was demonstrated to result in impaired T cell immunity [80].

However, this finding was suggested to be the result of dysregulated production of type I

interferon as no defect was found in LPS-induced NFκB activation or IL-12 production.

Together these findings suggest that IKK2 may play a dominant role in TLR-triggered activation

of NFκB in DCs.

IKK1 and IKK2 both contain two serines residues whose phosphorylation is a

prerequisite of IKK activation [81-83]. However, the identity of the kinase that phosphorylates

these residues remains controversial [69]. There have been suggestions that the IKKs may auto-

phosphorylate, however the recently described crystal structure of IKK2 contests this possibility

[84]. The kinase TAK1, in association with adaptors TAB2 and TAB3, is often suggested to

phosphorylate IKK1 and IKK2 downstream of TLRs and cytokine receptors [85, 86]. Activation

of these receptors results in K63-linked polyubiquitination of TRAF6. These ubiquitin chains are

required for the recruitment and activation of TAK1 mediated phosphorylation of the IKK

complex [85]. In support of a role for TAK1 directly phosphorylating IKK1 and IKK2, it was

found that deletion of the TAK1 kinase domain results in impaired IKK activation [86].

However, constitutively activate TAK1 is insufficient to activate IKK [85]. Furthermore, the

dependence of NFκB activation on TAK1 appears to be cell type specific [87]. TAK1-deficiency

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in DCs results in the disruption of DC and myeloid homeostasis and an impaired ability to prime

T cell responses, demonstrating its important role in DC biology [88].

The kinase RIP1 has also been suggested to phosphorylate the IKK complex. RIP1 is

required for NFκB activation in response to TNFα and its overexpression promotes spontaneous

IKK activity [58, 89]. However, catalytically inactive mutant RIP1 is able to support NFκB

signaling, demonstrating that it is unlikely to directly phosphorylate IKK1 and IKK2 [58]. RIP1

is polyubiquitinated during NFκB signaling, suggesting that it may act through recruitment of

NEMO or kinases in order to facilitate the phosphorylation of the IKK complex.

Sequestration and release by IκB proteins

The inhibitor of κB (IκB) proteins, as their name suggests, were first identified as inhibitors of

NFκB activation. By virtue of their multiple ankyrin repeat domains (ARDs), IκBs bind to NFκB

dimers and regulate their activation. IκBs may be categorized into three groups: the classical

IκBs (IκBα, IκBβ, and IκBε), the precursor IκBs (p105 and p100), and atypical IκBs (IκBNS,

IκBζ, IκBη, and Bcl-3) [90]. The classical and precursor IκB are constitutively expressed and act

primarily through cytoplasmic sequestration of NFκB dimers. The atypical IκBs are

predominantly localized to the nucleus and have diverse functions in the regulation of NFκB.

Furthermore, the expression of most atypical IκBs is low in unstimulated cells and induced upon

NFκB activation, suggesting that their biology is distinct from classical and precursor IκBs.

Where not explicitly stated otherwise, our discussion of IκB proteins refers specifically to the

classical IκB members.

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IκBα is the most thoroughly studied of the classical IκB proteins. While early studies

focused on IκBα, many properties identified have been found to be shared amongst the IκB

proteins. Following the activation of the IKK complex, IκB proteins are phosphorylated on two

key serine residues. This phosphorylation allows IκB proteins to be detected by β-transducin

repeats-containing protein (βTrCP) which acts as a substrate recognition subunit of the Skp,

Cullin, F-box-containing complex (SCF) E3 ligase complex [91]. βTrCP-SCF proceeds to

catalyze the addition of a K48-linked polyubiquitin chain on IκB, leading to its proteasome-

mediated degradation. NFκB dimers are thereby released from IκB inhibition and translocate to

the nucleus. While this mechanism is classically thought to act to inhibit NFκB, it also ensures

stimulus-responsiveness. Mouse embryonic fibroblasts deleted for IκBα, IκBβ, and IκBε have an

increase in nuclear p65, resulting in mild constitutive NFκB activation [92]. However, the

majority of p65 remained in the cytoplasm in association with p100 and p105, demonstrating

their ability to act as typical IκB proteins. Furthermore, IκB-deficient cells were found to be

refractory to TNFα-induced NFκB activation. These findings suggest that the formation of

NFκB:IκB complexes, in addition to its role in preventing spontaneous activation, is required to

respond to NFκB-activating stimuli.

While the classical IκB proteins share many core features, they are not functionally

redundant. This is clearly demonstrated by the individual phenotypes of IκB knockout mice.

Deletion of IκBα results in severe dermatosis and granulopoiesis, and as a consequence, death at

7-10 days of age [93]. IκBα-deficiency is additionally found to result in sustained NFκB

activation following treatment with TNFα or LPS, solidifying the role of IκBα in restricting

canonical signaling. The phenotypes of IκBβ and IκBε knockouts are comparatively mild, with

minor defects in specific immune populations, cytokine production, and the balance of antibody

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isotypes [92, 94, 95]. While each IκB protein does exhibit unique biochemical properties, it

appears that the dramatic differences in these phenotypes may be largely due to differential

regulation of IκBα expression. This was demonstrated by the creation of an IκBα-deficient

mouse with IκBβ knocked-in under the control of the IκBα promoter [96]. Unlike IκBα knockout

mice, these IκBβ knock-in mice survived and displayed no obvious immune defects. Therefore,

the dramatic phenotype of IκBα-deficient mice is likely a consequence of the IκBα promoter

which is strongly induced by NFκB activation, thereby creating a negative feedback loop.

Each IκB proteins also displays preferential binding of different NFκB dimers. IκBα and

p105, for example, preferentially bind to p65-, p50-, and cRel-containing heterodimers while

IκBβ and IκBε are mostly found in association with p65 and cRel. Pioneering work, primarily

using fibroblasts, suggested that RelB is not controlled by the classical IκBs, and that it was

instead regulated primarily by p100 as a component of non-canonical NFκB activation [97].

However, recent work in DCs has demonstrated that IκBα binds to p50:RelB dimers in the

cytoplasm of unstimulated cells [98]. Furthermore, RelB was required for cytokine production in

response to the canonical NFκB stimulus CpG. These data suggest that in DCs, classical IκBs

may regulate RelB-containing dimers, thereby blurring the lines between canonical and non-

canonical signaling.

Modulation of transcription by NFκB dimers

NFκB transcription factors have a well-appreciated role in driving the expression of

inflammatory genes. Many of these are pertinent to DC biology, including CD40, CD80, CD86,

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IL-1β, IL-6, IL-12, TNFα, and CCR7. While the importance of NFκB is clear, the individual

contributions of the different NFκB proteins has not been fully elucidated.

Dimerization of the 5 NFκB proteins can result in up to 15 distinct homo- and

heterodimers, although 3 of these are not thought to bind to DNA [65]. The functional properties

as well as target specificity of these dimers is determined by their individual constituents. While

all NFκB proteins may bind to κB sites within DNA, individual proteins have distinct consensus

sequences, resulting in preferential binding of NFκB dimers to specific promoters [99].The

RelA, RelB, and cRel subunits all contain C-terminal transactivation domains. Dimers containing

these proteins can therefore directly drive activation of transcription. NFκB may enable

transcription through the direct recruitment of the p300/CBP transcriptional co-activator.

However, only some NFκB target genes appear to be regulated through direct p300/CBP

recruitment [100]. Mutation of a key phosphorylated serine residue in RelA abolishes its ability

to bind to p300/BCP while only affecting a subset of target genes [101]. Phosphorylation of

NFκB subunits may therefore contribute to the specificity of transcriptional activation.

As p50 and p52 lack TADs, p50:p50, p52:p50, and p52:p52 dimers are unable to directly

drive transcription. However, the lack of TADs in these dimers not preclude their ability to

control gene expression as they have been shown to both positively and negatively regulate

transcription [65]. This idea has been primarily explored in the context of p50 homodimers.

These homodimers may bind to consensus κB sites within DNA, thereby competing with

activating NFκB dimers. Indeed, unstimulated cells contain DNA-bound p50 homodimers which

upon stimulation, are replaced with p65- or cRel-containing dimers. Furthermore, upon DNA

binding, p50 homodimers have been shown to recruit histone deacetylases (HDACs) which

induce chromatin remodeling to inhibit the initiation of transcription [102]. It has also been

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suggested that the binding of p50 homodimers is not limited to κB sites. Interferon response

elements (IREs) are DNA motifs which bind IRF proteins in order to mediate both the

production and response to type I interferons. One study has demonstrated that p50 homodimers

may bind to IREs and thereby repress the binding of IRF proteins [103]. Furthermore, they

observed that the loss of Nfkb1, and therefore p50 and p105, results in increased IRF-binding and

IFNβ production in macrophages. In summary, NFκB dimers lacking TAD, namely those

exclusively comprised of p50 and p52 repress gene expression by inhibiting the binding of

activating NFκB dimers.

Homodimers of p50 and p52 have also been suggested to regulate transcription through

the recruitment of the atypical IκB protein B cell lymphoma-encoded protein 3 (Bcl3). Although

Bcl3 contains the multiple ARD of IκB proteins, its localization is primarily nuclear and it shows

preferential binding to p50 and p52 homodimers [104-106]. Several reports suggested that Bcl3

promotes the displacement of p50 and p52 homodimers from DNA, thereby relieving cells of

their transcriptional repression [107-109]. Additional studies have proposed that Bcl3 acts as a

co-activator of transcription for p50 and p52 homodimers [110, 111]. Indeed, Bcl3-deficient

macrophages have an impaired ability to produce the immunoregulatory cytokine interleukin 10

(IL-10) [112]. Alternatively, Bcl3 may also inhibit transcription of pro-inflammatory genes

through stabilization of DNA-bound p50 homodimers. The importance of this mechanism is

suggested by the hypersensitivity of Bcl3-deficient mice to septic shock [113]. Furthermore,

Bcl3 knockout macrophages were shown to produce elevated levels of TNFα and IL-1β in

response to LPS [114]. In summary, though modulation of the expression of pro- and anti-

inflammatory genes, Bcl3 conspires with p50 and p52 homodimers to repress inflammation.

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Termination of NFκB signaling

Strict regulation of NFκB is not only required to prevent inappropriate activation and swift

responses to stimuli, but also to terminate NFκB signaling and thereby prevent excessive

inflammatory responses. The most thoroughly studied mechanism of limiting NFκB activation is

the negative feedback loop provided by classical IκB proteins. Recent work has additionally

identified a role for targeted protein degradation in the termination of NFκB activation. A third

major mechanism limiting NFκB is through the regulation of ubiquitination of NFκB signaling

intermediates. The importance of these mechanisms is clearly illustrated by the severe

inflammatory phenotypes that arise from their dysregulation.

The dynamics of each classical IκB protein following canonical NFκB signaling is

unique. IκBα is rapidly degraded following IKK activation [90]. However, its transcription is

strongly induced by NFκB dimers, and the synthesis of new IκBα is therefore greatly increased

following NFκB activation [115-118]. This creates an auto-inhibitory loop in which newly

synthesized IκBα may inhibit NFκB activation. Following its synthesis, IκBα translocates to the

nucleus where it is able to target DNA-bound NFκB dimers. Indeed, IκBα actively promotes the

dissociation of NFκB dimers from DNA [119]. Furthermore, IκBα-NFκB complexes are more

tightly bound than DNA-NFκB complexes [120]. Together these studies suggest that IκBα

terminates NFκB signaling first by dissociating NFκB dimers from DNA and subsequently

binding to NFκB in order to prevent its re-association with DNA. IκBα also promotes the nuclear

export of IκBα-NFκB through its nuclear export sequence (NES) and through partial masking of

nuclear localization sequences (NLS) in NFκB dimers [121-123]. However, in contrast to the

embryonic lethality of IκBα-deficient mice, the phenotype of mice harboring a mutated IκBα

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NES is mild, characterized by abnormal B cell responses [124]. This finding suggests that the

dissociation of NFκB dimers from DNA, and not their subsequent nuclear export, is the most

important mechanism by which IκBα terminates NFκB signaling. In comparison to IκBα, both

the degradation and production of IκBε is slow [125]. However, newly synthesized IκBε may

still enter the nucleus and inhibit NFκB [126] . The relatively stable expression of IκBε is

thought to counteract the rapid oscillations of IκBα degradation and synthesis thereby stabilizing

gene expression.

Upon canonical NFκB signaling, IκBβ is also degraded, but with delayed kinetics in

comparison to IκBα. Although its promoter does contain a κB site and stimulation-induced

upregulation is observed, the expression of IκBβ is not controlled by NFκB [127]. IκBβ

expressed in unstimulated cells is constitutively phosphorylated on two serine residues that are

important for its inhibitor function [128]. Following cell stimulation, newly synthesized IκBβ

primarily exists in a form that lacks phosphorylation on these two serine residues. This

hypophosphorylated IκBβ may enter the nucleus and form stable complexes with DNA-bound

NFκB [129]. It has been subsequently suggested that these complexes stabilize DNA binding and

thereby prolong NFκB activation [130, 131]. This mechanism has been suggested to explain why

the production of inflammatory cytokines such as TNFα and IL-1β is enhanced by IκBβ as well

as the resistance of IκBβ knockout mice to septic shock and collagen-induced arthritis [95, 132].

In summary, following the activation of NFκB, unlike IκBα and IκBε, IκBβ does not inhibit

NFκB and instead promotes its sustained activity.

Termination of NFκB activation is also facilitated through targeted degradation of DNA-

bound NFκB dimers. Early evidence for this mechanism came from observations that inhibition

of the proteasome following TNFα treatment resulted in sustained NFκB occupancy and

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transcription [133]. It has been suggested that IKK1, primarily appreciated for its role in

initiating canonical and non-canonical signaling, may promote degradation of nuclear NFκB

[134, 135]. IKK1 phosphorylates a serine residue within RelA which leads to its proteasome-

mediated degradation. Additionally, several E3 ubiquitin ligases have been reported to target

DNA-bound NFκB. PDZ and LIM domain-2 Mystique (PDLIM2) is a ubiquitin ligase which has

been demonstrated to target nuclear p65 for degradation in macrophage [136]. Its deficiency

resulted in prolonged NFκB signaling and exaggerated cytokine production. Additionally, copper

metabolism domain-containing-1 (COMMD1) has been suggested to act as an adaptor protein,

recruiting a suppressor of cytokine signaling-1 (Socs1) E3 ligase complex to DNA-bound NFκB

[137]. Therefore, NFκB-mediated transcription is terminated through mechanisms that both

remove NFκB dimers from DNA and those that promote their degradation.

Regulation through ubiquitination

Ubiquitin is a 76 amino acid protein that is pervasively employed in the regulation of eukaryotic

life [138, 139]. Proteins may be covalently tagged with ubiquitin with resulting consequences for

their function, localization, or degradation. As with other post-translational modification such as

phosphorylation, both the addition and removal of ubiquitin from proteins is the result of the

tightly regulated and targeted activities of enzymes. Through a series of enzymatic reactions

involving ubiquitin-activating enzymes (E1), ubiquitin-conjugating (E2) enzymes, and ubiquitin-

ligating (E3) enzymes, ubiquitin is covalently added to lysine residues within proteins. Ubiquitin

itself has 7 lysine residues which facilitate further addition of ubiquitin, forming polyubiquitin

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chains. Alternatively, linear polyubiquitin chains may be formed through linkage through the N-

terminal methionine. The consequences of polyubiquitination are dictated by which residue

within ubiquitin is used for polymerization. For example, polyubiquitin chains linked through

lysine 48 (K48) are generally thought to promote proteasome-mediated degradation of targeted

proteins, while lysine 63 (K63) linked chains form scaffolds that act to recruit signaling

mediators. Polyubiquitin chains may be detected by other proteins through their ubiquitin-

binding domains (UBDs). Distinct UBD-containing proteins preferentially bind to differentially-

linked ubiquitin chains, thereby connecting the choice of lysine residue and downstream

function.

The formation of both K48- and K63-linked polyubiquitin chains plays pivotal roles in

the activation of NFκB. As previously described, the degradation of IκB proteins is dependent

upon the addition of K48-linked ubiquitin chains through the βTrCP-SCF E3 ligase complex.

Furthermore, experiments conducted with mutant ubiquitin have demonstrated that the formation

of K63-linked chains is critical for the activation of the IKK complex in response to IL-1β [140].

Additionally, recent evidence suggests that linear ubiquitin chains, whose addition is catalyzed

by the linear ubiquitin chain assembly complex (LUBAC), on these signaling intermediates may

also facilitate NFκB signaling [141]. Following TLR stimulation, ubiquitin chains are catalyzed

on multiple signaling intermediates including IRAK1, TRAF6, and NEMO. Indeed,

polyubiquitination of IRAK and TRAF6 has been demonstrated to be critical for TLR-induced

NFκB activation [142, 143]. The polyubiquitin chains act as scaffolds to which the UBDs of the

regulatory subunits TAB2 and NEMO bind, resulting in the recruitment of the TAK1 and IKK

complexes, respectively. The co-localization of these complexes is believed to facilitate the

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phosphorylation of both the TAK1 and IKK complexes by TAK1, thereby inducing the

degradation of IκBs.

TRAF6, in conjunction with the E2 enzyme complex composed of ubiquitin-conjugating

enzyme 13 (Ubc13) and ubiquitin-conjugating enzyme variant 1A (UEV1A), has been suggested

to be the E3 ligase which catalyzes the addition of K63-linked polyubiquitin chains to NFκB

signaling intermediates following TLR stimulation. The vital importance of this E3 ligase was

illustrated by the severely impaired NFκB signaling observed in TRAF6-deficient cells in

response to various canonical stimuli including TLR ligands [144, 145]. However, the nature of

TRAF6’s contribution to NFκB signaling is contentious with reports both supporting and

opposing a role for its ubiquitin ligating enzymatic domain [146-149].

The addition of polyubiquitin chains can be counteracted through the activities of

deubiquitinases (DUBs). These enzymes catalyze the hydrolysis of ubiquitin chains from

proteins and are therefore critical regulators of ubiquitin-mediated signaling. Several DUBs have

been directly implicated in the regulation of NFκB signaling, with A20 and cylindromatosis

(CYLD) being the most thoroughly studied. CYLD is a member of the ubiquitin-specific

protease (USP) family of DUBs and was originally identified as a tumour suppressor [150].

Subsequent work demonstrated a role for CYLD in negatively regulating NFκB signaling by

deubiquitinating signaling intermediates [151-153]. Targets of the DUB activity of CYLD have

been suggested to include TRAF2, TRAF6, NEMO, TAK1, and RIP1 [151-156]. Furthermore, it

was found that CYLD preferentially cleaves K63-linked ubiquitin chains, providing insight into

its ability to inhibit signaling [157]. The importance of CYLD in regulating pro-inflammatory

NFκB signaling was demonstrated by the development of an inflammatory bowel disease-like

pathology in CYLD-deficient mice [154]. In macrophages, the loss of CYLD was shown to

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directly impact TLR and TNFR signaling, resulting in enhanced activation of NFκB [158]. In

summary, the activities of ubiquitin ligases are required for activation of NFκB while DUBs act

to counter ubiquitination, thereby inhibiting NFκB.

Regulation through A20

A20, also known as tumour necrosis factor α-induced protein 3 (TNFAIP3), is a potent negative

regulatory of inflammation. Through its ability to modulate ubiquitination, A20 antagonizes

NFκB signaling downstream of PRRs, B cell and T cell antigen receptors, TNFα, IL-1β, and

CD40 [159-162]. The key role of A20 in limiting inflammation is illustrated by both the

phenotype of A20-deficient mice, which succumb to multiorgan inflammation and

autoimmunity, and the associations between A20 polymorphisms and multiple autoimmune

diseases [163, 164].

The biology of A20 is unique in that it is capable of catalyzing both the addition and

removal of polyubiquitin chains. The N-terminus of A20 contains a catalytic ovarian tumour

(OTU) domain which endows A20 with the ability to function as a DUB [160]. In comparison to

the more common USP-containing DUBs which frequently do not discriminate between different

ubiquitin linkages, OTU-containing DUBs are generally specific for one or several linkages

[165]. The OTU domain of A20 cleaves K63-linked ubiquitin in vivo [160, 166, 167]. The C-

terminus of A20 contains 7 zinc finger (ZnF) domains which are thought to additionally

contribute to the ability of A20 to regulate ubiquitination. ZnF4 has E3 ubiquitin ligase activity

and promotes the K48-linked polyubiquitination of targets proteins [161]. Additionally, both

ZnF4 and ZnF7 facilitate direct binding of A20 to polyubiquitin chains. Zn4 has been

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demonstrated to promote the interaction of A20 with ubiquitinated E2 enzymes [168], while

ZnF7 was reported to interact with linear ubiquitin chains on the signaling intermediates NEMO

[169]. A20 has also been suggested to collaborate with other ubiquitin-binding proteins. A20-

binding inhibitor of NFκB (ABIN) and Tax1-binding protein 1 (TAX1BP1) have been reported

to facilitate A20 function by acting as adaptor proteins between A20 and polyubiquitin chains

[170-172].

Both the E3 and DUB activities of A20 are thought to contribute to its ability to regulate

NFκB signaling. A20 negatively regulates signaling from TLRs through cleavage of K63-linked

polyubiquitin chains from TRAF6 [160, 173, 174]. Other identified targets of the DUB activities

of A20 include RIP1, following TNFα signaling [161], and RIP2, following NOD2 activation

[175]. As K63-linked polyubiquitination is required to transduce signaling in these pathways,

these cleavage events are thought to limit the activation of NFκB.

The ZnF4 domain of A20 catalyzes the addition of K48-linked polyubiquitin chains to

NFκB signaling intermediates, thereby promoting their degradation and inhibiting signaling. The

first identified target of the ZnF4 domain was RIP1 [161]. This finding suggested that A20

regulation of TNFα-induced RIP1 activation is two-fold; A20 catalyzes both the removal of K63-

linked chains and the addition of K48-linked chains, thereby limiting signaling and inducing

proteasome-mediated degradation of RIP1. Other identified targets of A20-induced K48

polyubiquitination include the E2 enzymes UBCH5 and UBC13 which are involved in the K63-

linked polyubiquitination of RIP1 and TRAF6, respectively [167]. Furthermore, the ZnF4

domain was further suggested to disrupt interactions between UBCH5 and UBC13 and the E3

ligases cIAP and TRAF6, respectively. Therefore, A20 inhibits the K63-linked

polyubiquitination of RIP1 and TRAF6 by disrupting and promoting the degradation of E2

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enzymes. ZnF7 has also been suggested to regulate NFκB signaling independently of any

catalytic activity. Linear ubiquitin chains attached to NEMO facilitate binding of A20 through

ZnF7, resulting in inhibition of NEMO activation [169].

Recent studies have aimed to determine the relative contributions of the E3 and DUB

activities of A20 in restricting inflammation. To address this issue, investigators generated two

A20 knockin mouse strains harboring mutations that rendered either the OTU domain or ZnF4

domain catalytically inert [176]. Both OTU and ZnF4 mutant strains lacked the severe and fatal

inflammation of A20-deficient mice, suggesting that neither the DUB nor the E3 enzymatic

activities of A20 are essential for its role in preserving immune homeostasis. However,

fibroblasts from both OTU and ZnF4 mutants displayed elevated NFκB activation following

treatment with TNFα. Co-expression of both mutant A20 molecules restored normal NFκB

signaling, demonstrating that these enzymatic domains can complement each other in trans. This

was suggested to be mediated through the homodimerization of A20 molecules. Subsequent

work confirmed that mice with an inactivated A20 OTU are grossly normal [177]. However, they

also reported normal NFκB signaling in response to TNFα and LPS. In summary, neither the

DUB nor the E3 functions of A20 are required to prevent the onset of fatal inflammatory

pathology. Furthermore, the roles of DUB and E3 activities in modulating NFκB activity may be

cell- or stimulus-specific. It may be that the functional loss of one catalytic domain may be

compensated by the other. Alternatively, the ability of A20 to regulate NFκB activation by

binding to polyubiquitinated proteins through ZnF4 and ZnF7 may be crucial for the preservation

of immune homeostasis.

The relevance of A20 to human disease has been demonstrated by multiple genome-wide

association studies (GWAS) which have correlated polymorphisms in the A20 locus with many

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autoimmune and inflammatory diseases including systemic lupus erythematosus (SLE),

inflammatory bowel disease (IBD), rheumatoid arthritis (RA), juvenile idiopathic arthritis, celiac

disease, Sjorgren’s syndrome, rheumatic heart disease, and systemic sclerosis, and coronary

artery disease. [164, 178]. A majority of these polymorphisms are within the promoter region of

A20, suggesting that regulation of A20 expression may govern the development of

autoimmunity. The expression of A20 is regulated by NFκB, thereby forming a negative

regulatory feedback loop for NFκB activation [179]. Therefore it may be that disruption of this

feedback loop supports the onset of autoimmunity. A recent report has identified DRE-antagonist

modulator (DREAM) as a negative regulator of A20 expression [180]. DREAM-deficient mice

have constitutive high expression of A20 and are resistant to LPS-induced lung pathology.

Together these studies suggest that the regulation of A20 expression is important for controlling

inflammation.

The associations between A20 polymorphisms and a wide range of autoimmune diseases

suggest that A20 has a pervasive role in maintaining tolerance. Indeed, A20 has been

demonstrated to have important cell-intrinsic roles across multiple lineages. Investigators

demonstrated that the fatal inflammation suffered by A20-deficient mice could be rescued

through the deletion of MyD88 or through administration of antibiotics, suggesting that A20 is a

key regulator of the innate sensing of microbiota through TLRs [173]. Conditional deletion of

A20 from macrophages and granulocytes results in the development of a RA-like disease

including polyarthritis, anti-collagen antibodies, and elevated IL-6 production, further

demonstrating the role of A20 in regulating innate immune cells [181]. Studies have also

identified cell-intrinsic roles for A20 in B cells, T cells, and intestinal epithelial cells [182-186].

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A20 has been demonstrated to be a key regulator of DC biology [187, 188]. DC-specific

ablation of A20 expression results in splenomegaly and lymphadenopathy driven by the

expansion of activated T cells and B cells, as well as autoimmune disease. A20-deficient DCs

were found to have a mature phenotype and produce elevated levels of inflammatory cytokines.

Furthermore, this phenotype was dependent upon MyD88, suggesting a key role for A20 in

restricting TLR signaling in DCs [187]. RNAi silencing of A20 in DCs has also been reported to

enhance T cell responses including those direct towards a tumour [189, 190]. Together these

studies demonstrate that A20 controls DC homeostasis through regulation of NFκB and thereby

limits their ability to generate adaptive immune responses.

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T cell activation

The mammalian immune system is endowed with the ability to respond to a vast array of

potential antigens. This capability is in part the product of the somatic recombination of T cell

receptors. While the random diversity generated by recombination allows the adaptive immune

system to respond to pathogen-derived antigens, it also leads to the generation of T cells that

may react against self or the innocuous environment. The ability of T cells to mount powerful

immune responses means that inappropriate T cell activation has severe consequence. The

selection of antigen-specificity and the activation of peripheral T cells are therefore both strictly

regulated. We will herein focus on key events during T cell activation and explore how these

events are controlled by essential regulatory molecules.

T cell Receptor Stimulation

The hallmark of a T cell response is the specificity inscribed within its T cell receptor (TCR).

The successful cloning of the antigen receptor, those chains which encode TCR specificity,

therefore laid the groundwork for understanding the molecular events governing T cell activation

[191-193]. The TCR is a multimeric complex which interacts with cognate antigen-MHC

complexes on antigen-bearing cells and initiates the transduction of signals which lead to T cell

activation. Antigen specificity of conventional T cells is determined by TCRα and TCRβ chains.

These two proteins are the product of the intricately regulated recombination and selection

events that occur during T cell development in the thymus. While TCRα and TCRβ dictate

specificity, they lack the capacity to induce intracellular signaling cascades. The TCRα and

TCRβ are found in association with various CD3 molecule dimers (CD3δε CD3γε, and CD3ζζ)

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which are crucial for signal transduction from the TCR [194]. Unlike TCRα and TCRβ, CD3

molecules contain long intracellular tails which enable them to propagate signaling. These

cytoplasmic tails contain immunoreceptor tyrosine-based activation motifs (ITAMs) which are

composed of an amino acid sequence characterized by critical tyrosine and leucine/isoleucine

residues flanking a spacer sequence. The activation induced phosphorylation of tyrosines within

these ITAMs form the basis of their signaling potential [195]. In sum, the TCR signaling

complex is composed of antigen-specifying TCRα and TCRβ chains and signaling propagating

CD3 moieties.

Signal transduction from the TCR is initiated upon ligation by an MHC-peptide complex

(Figure I-4). While the precise mechanism of the initiation of TCR activation remains

controversial [194], the consequence of this interaction is the phosphorylation of the tyrosine

residues within the CD3 ITAMs by members of the Src family of kinases, such as Lck or Fyn.

These phosphorylated ITAMs then act to recruit a proximal signaling complex. The first protein

recruited to this complex is ζ-associated protein of 70 kilodaltons (Zap70). TCR signaling is

propagated through Zap70 by virtue of its protein tyrosine kinase domain which phosphorylates

two primary targets, linker of activation of T cells (Lat) and Src homology 2 domain-containing

leukocyte phosphoprotein of 76 kilodaltons (SLP76). The phosphorylation of Lat and SLP76 and

their recruitment into the proximal TCR signaling complex is fundamental for T cell activation;

TCR signals are greatly impaired in their absence [193, 196, 197]. The formation of this

proximal signaling complex composed of Zap70, SLP76, and Lat in association with the TCR is

also a point of divergence for T cell signaling. From this node, several signaling cascades

emerge, leading to the activation of multiple transcription factors which drive the activation of T

cells.

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Figure I-4. T cell receptor engagement initiates a cascade of signaling events. Upon ligation

of the TCR with a cognate MHC-antigen complex, Src family kinases such as Lck, in association

with CD4 or CD8 molecules, are recruited to the site of TCR engagement. Lck phosphorylates

ITAM motifs contained within intracellular domains of CD3 proteins. Through its Sh2 domains,

Zap70 is recruited to these phosphorylated ITAMs and promotes the formation of a proximal

signaling complex through the recruitment of LAT, PLCγ1, Grb2, SOS, and Slp76. PLCγ1

catalyzes the production of the lipid signaling intermediates DAG and IP3. The production of

DAG leads to activation of PKCθ, which along with SOS, promotes the activation of MAPKs

and consequently the activation of AP1 family transcription factors. PKCθ additionally activates

the BCM complex which leads to activation of NFκB transcription factors. The release of IP3

promotes the secretion of Ca2+

cations from the ER which in turn promotes an influx of

extracellular Ca2+

cations. The resulting increase in Ca2+

concentration promotes the activation of

calmodulin and calcineurinin which leads to the dephosphorylation and nuclear translocation of

NFAT.

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The phospholipase C γ1 (PLCγ1) is subsequently recruited into the proximal signaling

complex. Kinases of the Tec family phosphorylate PLCγ1, activating its enzymatic activity.

PLCγ1 targets phosphatidylinositol (4,5)-bisphosphate (PI(4,5)P2) contained within the inner

leaflet of the plasma membrane. The hydrolyzation of PI(4,5)P by PLCγ1 produces

diacylglycerol (DAG) and inositol triphosphate (IP3), two signaling intermediates which go on to

further propagate signals from the TCR.

The release of IP3 from the plasma membrane results in ligation of IP3 receptors

expressed on the endoplasmic reticulum. This interaction leads to the efflux of Ca2+

cations from

the endoplasmic reticulum. This release ultimately leads to an influx of extracellular Ca2+

cations

and consequently, a rise in the concentration of intracellular Ca2+

. The calcium-sensitive protein

calmodulin is then activated, enabling it to activate several downstream signaling intermediates.

The most widely appreciated effect of calmodulin activation in T cells is the induction of a

conformational change in calcineurin, activating its phosphatase activity. Calcineurin then

dephosphorylates NFAT, allowing it to translocate into the nucleus and where it coordinates with

other transcription factors to drive expression of target genes such as IL-2.

The other product of PLCγ1 activation, DAG, activates several downstream signaling

intermediates including protein kinase C θ (PKCθ). CARD-containing membrane-associated

guanylate kinase protein 1 (Carma1) is subsequently phosphorylated by PKCθ. Consequently,

Carma1 forms a trimolecular complex with mucosa-associated lymphoid tissue lymphoma

translocation gene 1 (Malt1) and Bcl10. The formation of this Carma1:Bcl10:Malt1 (CBM)

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complex leads to the activation of IKK, and consequently, the activation and translocation of

NFκB transcription factors [198].

An additional component of TCR signaling is the Ras-mediated activation of AP-1

transcription factor. This is mediated through at least two distinct pathways. Firstly, PLCγ1-

produced DAG activates a guanine exchange factor (GEF) protein that leads to the activation of

Ras. Secondly, the adaptor protein Grb2 is recruited to Lat in the TCR proximal signaling

complex. Grb2 is associated with an additional GEF protein, further promoting the activation of

Ras. Activated Ras initiates a phosphorylation cascade involving mitogen-activated protein

kinases (MAPKs) such as extracellular signal-regulated kinase (Erk) ultimately resulting in the

activation of AP-1.

The consequence of TCR engagement is the initiation of multiple signaling cascades that

promote T cell activation. Some functions of TCR stimulation are carried by dynamic regulation

of cellular proteins, such as the cytoskeletal rearrangements that accompany T cell activation.

However, the major consequence of TCR stimulation is the activation of multiple transcription

factors, including NFAT, NFκB, and AP-1, which mediate T cell proliferation and activation.

Co-regulation

It has long been suggested that stimulation through the TCR alone is insufficient to induce robust

T cell activation. This idea led to the development of a “two signal” model of T cell stimulation

which postulates that a second signal must accompany TCR engagement in order for activation

to occur [199]. This theory was supported by the discovery of CD28, a molecule whose

stimulation in the context of TCR ligation led to enhanced Il-2 production, proliferation, and cell

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survival [200]. Since the discovery of CD28, our understanding of T cell co-stimulation has

grown considerably; dozens of molecules, both co-stimulatory, those who support T cell

activation, and co-inhibitory, those which dampen T cell activation, have been identified. These

molecules are primarily of the Ig-superfamily or the tumour necrosis factor superfamily

(TNFSF). While the basic idea of an induced stimulus being required still stands, we now

appreciate that T cell responses are not dictated by a single “second signal”. Instead, T cell

activation is the summation of the numerous interactions between the many co-stimulatory

molecules, co-inhibitory molecules, and cytokines expressed by T cells, APCs, and the

microenvironement. DC maturation, therefore, is not simply the induced expression of a “second

signal”, but the adaptation of an expression profile including many co-stimulatory molecules and

cytokines which help dictate the magnitude and differentiation of a T cell response.

CD28 remains the most well characterized T cell co-stimulatory molecule. A member of

the Ig superfamily, CD28 is constitutively expression on naïve T cells. Its ligands, CD80 (B7.1)

and CD86 (B7.2), are inducibly expressed on the surface of antigen presenting cells. During T

cell activation, ligation of CD28 is able to reinforce TCR signaling cascades. The cytoplasmic

tail of CD28 is able to recruit multiple proteins by which signals are propagated. One such

molecule is phosphatidylinositide 3-kinase (PI3K) which catalyzes the production of

phosphatidylinositol 3,4,5-triphosphate (PIP3) leading to the activation of Akt. The effects of Akt

activation are broad and include enhancing NFAT activity and promoting assembly of the CBM

complex, thereby enhancing TCR induced NFκB activation [201]. However, the role of PI3K in

mediating the effects of CD28 co-stimulation has been challenged by recent studies that

demonstrated through targeted mutagenesis of CD28 that the recruitment of PI3K is not essential

for the function of CD28 [202, 203]. CD28 can also associate with Grb2 to promote MAPK and

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AP-1 activation which may additionally contribute to T cell activation. Through these effects and

more, co-stimulation through CD28 enhances T cell proliferation and survival.

CD28 is not the sole Ig-superfamily molecule to modulate T cell activation. Surface

expression of cytotoxic T lymphocyte antigen-4 (CTLA-4) is induced upon T cell activation

where it is believed to dampen T cell responses [204]. CTLA-4 has high affinity for the CD28

ligands CD80 and CD86 and can therefore bind to them on the surface of APCs. Several distinct

models of CTLA-4-mediated suppression of T cell responses have been proposed including

competition for CD80/CD86, suppression of APCs, transendocytosis of CD80/CD86, and

through regulatory T cells [204, 205]. Regardless of the mechanism, the importance of CTLA-4

in regulating T cell responses was clearly demonstrated by the fatal inflammation suffered by

CTLA-4-deficient mice [206, 207]. Thus T cell activation can both be supported and suppressed

by cell surface molecules.

While many co-stimulatory molecules of the Ig and TNF superfamilies have been

demonstrated to play an important role during T cell activation, none appears to be an absolute

requirement for T cell activation. For example, while CD28 is required for normal immune

responses against vesicular stomatitis virus (VSV) [208], CD8+ T cell responses against

lymphocytic choriomeningitis virus (LCMV) and murine gamma herpesvirus are normal in the

absence of CD28 [209]. Likewise, mice deficient in the TNFSF co-stimulatory ligand 4-1BBL

mount normal anti-LCMV responses while displaying impaired immunity to influenza [210].

These observations suggest that the requirement for co-stimulation is context dependent.

T cell responses are regulated, in part, through crucial signaling events that are triggered

by ligation of their antigen receptor, as well as co-stimulatory and co-inhibitory receptors. While

loss of core TCR signaling components has catastrophic consequences for T cell development

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and activation, and loss of important co-inhibitory molecules such as CTLA-4 or programmed

death 1 (PD1) leads to the development of autoimmunity [206, 207, 211], the loss of co-

stimulatory molecules does not necessarily have obvious or severe consequences. However, the

importance of co-stimulatory molecules should not be understated, as in the context of a specific

immune response their effects can be profound. Understanding the regulation and downstream

signaling invoked by these molecules is therefore paramount to understanding immune responses

in the array of contexts in which they arise.

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The regulation of T cell activation by phosphorylation

Phosphorylation is a dynamic process that ubiquitously regulates all aspects of cellular life.

Dynamic regulation of phosphorylation is essential for both the maintenance of immune

homeostasis and for mounting powerful immune responses. Leukocytes express more protein

tyrosine kinases (PTKs), molecules that add phosphate to tyrosine residues, and protein tyrosine

phosphatases (PTPs), molecules that hydrolyze phosphate from tyrosine residues, than any other

mammalian cell type save for neurons [212]. This observation likely reflects the ability of the

immune system to rapidly and dramatically respond to its environment. The activation of T cells

is fundamentally regulated by phosphorylation. Protein phosphorylation is often viewed as

activating and dephosphorylation as dampening. However, the truth is often much more nuanced.

While we have already touched on some key phosphorylation events that propagate signals from

the TCR, we will herein discuss how T cell activation is dynamically regulated by competing

activities of kinases and phosphatases.

Kinases

Kinases are molecules whose enzymatic activity adds phosphate groups to other molecules. They

are broadly characterized by the kind of molecule they phosphorylate, with the most common

options being lipids, serine/threonine residues, or tyrosine residues. Approximately 2% of the

human genome is comprised of kinases, underscoring their universal importance [213]. Many of

the fundamental TCR signaling molecules already discussed, including Zap70, Lck, and Fyn are

PTKs. These molecules are critical for TCR activation. For example, loss of Zap70 [214, 215] or

combined loss of the Src family kinases Lck and Fyn[216] [217] completely abolishes signaling

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through the TCR, leading to arrested T cell development. PTKs are normally thought to

positively influence signaling and cell activation. However, PTKs may also negatively regulate T

cell activation. Carboxy-terminal src kinase (Csk) is known to phosphorylate Lck, an event

which inhibits its activation. In the absence of Csk, Lck is constitutively active, leading to

dysregulated TCR signaling [218]. Therefore, PTKs may play both positive and negative roles in

controlling T cell activation.

PTKs may also play more nuanced roles in regulating T cell activation. Inducible T cell

kinase (Itk) is a member of the Tec family of PTKs [219]. The production of PIP3 by PI3K

recruits ITK to the plasma membrane following TCR stimulation. Upon its arrival, Itk binds to

phosphorylated residues on Slp76 and phosphorylates PLCγ1. Full activation of PLCγ1 requires

Itk-mediated phosphorylation; loss of Itk results in dampened Ca2+

mobilization and Ras

activation [220]. However, in comparison to the loss of the core kinases, TCR stimulation in the

absence of Itk is merely dampened and not eliminated. TCR signaling is not a binary event and

the refined modulations provided by a regulatory molecule such as Itk can therefore influence the

nature of a T cell response. The differentiation of CD4+ T cells is thought to be largely regulated

by the cytokine milieu. However, the nature of TCR signaling is also believed to influence

lineage decisions, with strong TCR signals promoting T helper 1 (Th1) differentiation and

weaker TCR signals promoting T helper 2 (Th2) differentiation [221]. Concordantly, loss of Itk

has been demonstrated to have a dramatic impact on CD4+ T cell differentiation. Itk-deficient

mice mount severely impaired Th2 responses during infection with Leishmania major or during

asthma induction [222, 223]. By contrast, Itk-deficient mice mount relatively normal Th1

responses [220]. Although the mechanism by which Itk specifically regulates Th2 immunity

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remains uncertain [220], these studies clearly demonstrate that PTKs can profoundly impact the

nature of a T cell response.

In summary, PTKs play fundamentally important roles in regulating the signaling

cascades leading to T cell activation. Kinases often drive activating signaling events. However,

they may also restrict T cell activation or act as fine-tuning mechanisms, controlling the strength

of TCR signaling or impacting T cell differentiation.

Phosphatases

Phosphorylation is a reversible process. While the enzymatic activities of kinases have been

thoroughly studied in T cells, comparatively little is known about the process of

dephosphorylation. An initial appreciation of the role of phosphatases in regulating T cell

biology came from experiments that demonstrated that treatment of activated T cells with

sodium orthovanadate, a broad inhibitor of PTKs, resulted in prolonged T cell activation and

proliferation. It was subsequently demonstrated that treatment of human T cells with the

phosphatase inhibitor pervanadate resulted in spontaneous T cell activation and IL-2 production

[224, 225]. Therefore phosphatases have critical roles in regulating T cell biology both during

activation and the steady state.

Like kinases, phosphatases can be broadly classified by their target substrate.

Phosphatases may exhibit specificity for phosphorylated lipids, tyrosine, histidine, serine and

threonine, or dual specificity for tyrosine and serine/threonine. Phosphatases of all types regulate

T cell biology. For example, Src homology-2 domain-containing inositol 5-phosphatase 1

(Ship1) and Phosphatase and tensin homolog (Pten) are two lipid phosphatase which counter the

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activities of PI3K by catalyzing the transformation of PI(3,4,5)P2 into PI(4,5)P2 or PI(3,4)P2,

respectively [226, 227]. However, we will herein focus on PTPs, those phosphatases which

dephosphorylate phosphotyrosine.

The function of PTPs should not be viewed as simply housekeeping required to reverse

the phosphorylation following the activation of PTKs. Indeed, the human genome encodes for

more PTPs than it does PTKs, underscoring the intricate regulation carried out by PTPs [213].

PTPs have been further categorized into 4 classes based on conserved catalytic elements shared

among PTPs [228]. Of these, Class I is by far the most abundant and is comprised of PTPs that

contain a conserved cysteine-based catalytic motif and have specificity for tyrosine or dual

specificity for tyrosine and serine/threonine. T cells express at least 60 PTP molecules [229].

While PTPs are often thought to universally be inhibitors of immune cell activation, this is often

not the case. Though the majority of PTPs in T cells do appear to be inhibitory, some PTPs can

function to promote T cell activation [212].

CD45 is the most thoroughly studied PTP in T cell biology, likely a reflection of the fact

that it actively promotes T cell activation. Indeed, the phosphatase activity of CD45 is required

for TCR signaling [230]. CD45 is a membrane bound PTP that acts by targeting the inhibitory

phosphotyrosine on Lck. In this way, the balance of the enzymatic activities of CD45, providing

activating dephosphorylation, and Csk, providing inhibitory phosphorylation, control the

activation of Lck and therefore TCR signaling.

PTPs often provide inhibitory regulation to counter the activities of PTKs in T cells. One

such phosphatase is Protein tyrosine phosphatase non-receptor type 22 (Ptpn22, also known as

Lyp or Pep) which negatively regulates TCR signaling. Ptpn22 has been demonstrated to interact

collaborate with Csk in order to inhibit Src family kinases such as Lck and Fyn [231]. Ptpn22

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catalyzes the removal of an activating phosphorylation from Lck, thereby inhibiting the initiation

of TCR signaling [232]. Studies of Ptpn22 deficiency helped illustrate the important role that

PTPs can play in regulating T cell biology. Ptpn22 knockout mice develop splenomegaly and

lymphadenopathy with an accumulation of effector T cells and elevated serum antibodies [233].

While immune homeostasis is clearly perturbed in Ptpn22 knockout mice, these manifestations

fall short of overt autoimmunity. Furthermore, the loss of Ptpn22 results in augmented TCR

signaling and Lck phosphorylation in effector-memory cells, but not naïve cells, highlighting the

complexity of PTP regulation [233].

The essential regulation provided by PTPs was demonstrated by GWAS studies that

identified Ptpn22 as one of the strongest loci outside of the MHCII loci to associate with the

development of autoimmune disease [234]. The single nucleotide polymorphism (SNP) C1858T

in Ptpn22 leads to a tryptophan to arginine substitution (R620W). This SNP has been found to be

associated with type 1 diabetes, systemic lupus erythematosus, myasthenia, gravis, Graves

disease, Addison’s disease, vitiligo, and juvenile idiopathic arthritis, among others [234].

Surprisingly, subsequent functional studies of the Ptpn22 risk allele have determined that

R620W is a gain-of-function mutation, with carriers of the mutation exhibiting impaired TCR

signaling [235-237]. How a gain-of-function mutation in an inhibitor of TCR signaling supports

the development of autoimmune disease remains unclear, although altered thymic selection and

regulatory T cell function have been proposed [238-240]. These finding clearly demonstrate that

Ptpn22, like most PTPs, is not simply an “off switch” for cell activation, but instead is an

intricate regulator of immune homeostasis and activation.

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Sh2 domain-containing phosphatases

There are two members of Sh2 domain-containing phosphatase family, namely Shp1

(PTPN6/HCP) and Shp2 (PTPN11) [241]. While these two molecules have conserved structures

and regulatory mechanisms, their biological roles are considerably distinct. Shp1 is expressed in

hematopoietic cell and is generally considered to be an attenuator of immune cell activation. By

contrast, Shp2 is ubiquitously expressed in mammalian cells and positively regulates cell

activation through control of MAPK signaling.

Shp proteins are non-transmembrane proteins that contain a single catalytic domain

[241]. This domain is a classical cysteine-based phosphatase domain making both Shp1 and

Shp2 Class I PTPs. The Shp proteins are named for their two Src-homology-2 (N-SH2 and C-

SH2) domains which are located within their N-terminus. Sh2 domains mediate interactions with

phosphorylated immunoreceptor tyrosine-based inhibitory motifs (ITIMs) or immunoreceptor

tyrosine-based switch motifs (ITSMs). ITIM motifs are found on many receptors expressed on

the surface of hematopoietic cells such as CD5, B- and T-lymphocyte attenuator (Btla),

Interluekin-4 receptor α (IL4Rα), Programmed death-1 (PD1), Carcinoembryonic antigen-related

cell adhesion molecule 1 (Ceacam1), single regulatory protein α (Sirpα), and numerous NK cell

receptors, among many more [242]. ITSM expression is much more limited but is also found on

BTLA and PD1 [242]. ITIM and ITSM motifs contain a single tyrosine residue which may be

phosphorylated, facilitating the recruitment of Shp1 and Shp2. However, not all ITIM motifs

equally recruit Shp1 and Shp2 [241]. Additionally, other Sh2-domain containing proteins such as

Ship1 and members of the Suppressor of cytokine signaling (Socs) family of ubiquitin ligases are

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also recruited to phosphorylated ITIM motifs. Understanding the role of a Sh2-containing protein

in regulating a given ITIM-containing protein is therefore non-trivial.

In addition to their role in facilitating intermolecular interactions with ITIM- and ITSM-

containing proteins, the Sh2 domains of Shp proteins are believed to contribute to their

regulation. Resolution of the Shp2 crystal structure and accompanying functional studies

demonstrated that in the basal state, Shp2 is folded in on itself such that its N-SH2 domain is

obstructing the catalytic PTP domain [243, 244]. It has been hypothesized that the binding of the

C-SH2 domain to phosphotyrosines may promote the unfolding and activation of Shp2

phosphatase activity [241]. Subsequent analysis of the structure of Shp1 strongly suggests that

analogous regulatory mechanisms also control its activity [245, 246].

The C-terminal regions of Shp1 and Shp2 also contribute to their regulation. Shp2, but

not Shp1, contains a C-terminal proline-rich region which may mediate intermolecular

interactions with SH3 domains. Although not contained in Shp1, a minor Shp1 splice variant,

Shp1L, also has a proline-rich region [247]. However, any functional significance of these

regions has not yet been demonstrated. It has also been suggested that Shp proteins may

themselves be regulated by phosphorylation. Both Shp1 and Shp2 contain two tyrosine residues

at their C-terminus. It has been proposed that phosphorylation of these residues increases the

activity of their PTP domains [248-250]. Shp1 contains two additional regulatory elements in its

C-terminus that are thought to control its localization. One of these, a nuclear localization signal,

has been demonstrated to promote accumulation of Shp1 in the nucleus following cytokine

stimulation [251]. The Shp1 C-terminus additionally contains a SKHKED motif which may

promote localization of Shp1 into lipid rafts [252, 253]. Moreover, this motif was found to be

necessary for the inhibitory function of Shp1 [253].

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While Shp1 and Shp2 share common structural and regulatory elements, their roles in

regulating immunity are largely divergent. Shp1 has been characterized as negatively regulating

the activation of a multitude of hematopoietic cell lineages. Shp2, however, is believed to

promote cellular activation, primarily through its ability to facilitate activation of MAPK

signaling cascades. While the mechanism by which Shp2 accomplishes this is still debated, most

cell types appear to require Shp2 for complete activation of Erk signaling.

Our understanding of the biological role of Shp2 in immune cells has been impaired by

the embryonic lethality of Shp2 deficiency in mice [254, 255]. Furthermore, experiments with

bone marrow chimeras demonstrated that Shp2 knockout cells cannot reconstitute B- and T-

lymphopoiesis in Rag2 deficient hosts, demonstrating the essential role of Shp2 in the

development of these lineages [256]. Consequently, the earliest experiments examining the role

of Shp2 in T cell biology utilized a dominant negative Shp2 containing a cysteine to serine (C/S)

mutation in its catalytic domain. Jurkat T cells expressing mutant Shp2 protein have dampened

Erk activation following TCR stimulation [257]. Accordingly, mice expressing Shp2 C/S in the

T-lineage accumulate activated T cells [258]. Subsequently, the generation of conditional Shp2

knockout mice revealed a clear role for Shp2 in mediating Erk signals during thymocyte

selection [259]. Conditional deletion of Shp2 from T cells has also been demonstrated to

promote B16 melanoma progression and metastasis, further demonstrating a positive role for

Shp2 in regulating T cell activation.

By virtue of its ITIM/ITSM binding SH2 domain, Shp2 may also propagate signaling

from inhibitory T cell receptors. The cytoplasmic tail of PD-1 contains both ITIM and ITSM

motifs which become phosphorylated upon its ligation. The ITSM of PD1, in particular, is able

to recruit both Shp1 and Shp2, although Shp2 appears to have a stronger association [260].

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Furthermore, stimulation of PD-1 with PD-1 ligand-2 (PD-L2) results in phosphorylation of

Shp2 [261]. Moreover, the introduction of the dominant negative Shp2 C/S mutant, but not a

Shp1 mutant, abolished the ability of PD-1 ligands to supress IL-2 production in T cells [262]. In

summary, Shp2 is a multipurpose regulator of T cell activation, promoting the activation of Erk

signals following TCR stimulation while also propagating the effects of inhibitory receptors such

as PD-1.

Regulation of inflammation by Shp1

Our understanding of the role that Shp1 plays in regulating immunity began with the discovery

of a spontaneous mutant mouse within the Jackson laboratories. These mutants, dubbed

“motheaten”, suffered from retarded growth, inflammatory skin lesions, myeloid hyperplasia,

hypergammaglobulinemia, interstitial pneumonia, and consequently, premature death with an

average life span of 3 weeks [263, 264]. Subsequently, another spontaneous mutant arose that

developed a milder motheaten phenotype. While still exhibiting many of the same pathological

manifestations including the development of interstitial pneumonia and inflammatory skin

lesions, these mutant mice survived for 9 weeks on average and were thusly named “viable

motheaten”. The recessive motheaten and motheaten viable alleles were both mapped to

chromosome 6. However the precise genetic etiology of the motheaten phenotype proved elusive

for two decades following the discovery of motheaten mice.

Seminal work from the laboratories of Florence Tsui and David Beier identified that the

motheaten phenotypes were the result of mutations within Ptpn6, the gene encoding for Shp1

[265, 266]. The motheaten allele was found to contain a single nucleotide deletion which led to

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aberrant splicing, premature termination of translation, and no functional Shp1 production [265].

The motheaten viable allele contained a substitution mutation within a donor splice site, leading

to the expression of two aberrantly spliced transcripts and ultimately the retention of 10-20% of

Shp1 catalytic activity [265]. These findings clearly established a critical role for Shp1 in

regulating inflammation. Importantly, this was also the first demonstration that the activities of

phosphatases are fundamental to the regulation of immunity.

The phenotypes of Shp1 mutant mice are severe, with abnormalities observed in all

lineages of immune cells. This multifaceted phenotype therefore complicated the analysis of

cell-intrinsic Shp1 function. The earliest studies used motheaten and motheaten viable cells, as

well as an engineered dominant negative Shp1 mutant (C453S), to decipher the role of Shp1in

regulating signaling in immune cells. These reports identified roles for Shp1 in many

hematopoietic lineages where it was found to negatively regulate signaling downstream of the B

cell receptor (BCR) [267, 268], killer cell inhibitory receptors (KIRs) [269], erythropoietin

receptor (EpoR) [270], macrophage colony stimulating factor (M-CSF) [271], granulocyte-

macrophage colony stimulating factor (GM-CSF) [272], Interluekin-3 receptor (IL-3R), as well

as integrin proteins [273].

More recently, the development of a floxed Ptpn6 allele has facilitated the elucidation of

cell-intrinsic functions of Shp1 [274]. Conditional deletion of Shp1 from B cells demonstrated

that Shp1 restricts the differentiation of B-1a cells, production of auto-reactive antibodies,

development of glomerulonephritis, while also promoting survival of memory B cells [274, 275].

Neutrophil-specific deletion of Shp1 results in the development of cutaneous inflammation due

to dysregulated integrin signaling [276]. The loss of Shp1 in DCs leads to the loss of immune

tolerance including the expansion of myeloid-, T-, and B-lineage cells in addition to the

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production of autoantibodies [276]. This phenotype was suggested to be caused by enhanced Erk

signaling downstream of TLRs. Therefore, in addition to its global role in preventing lethal

inflammation, Shp1 has clear and distinct cell-intrinsic functions.

Regulation of T cells by Shp1

The motheaten phenotype is independent of both B cells and T cells [277]. However, T

cells from motheaten mice exhibited enhanced proliferation in response to TCR signaling,

inspiring a series of studies examining the role of Shp1 in regulating T cell activation [278].

Many of these investigations described a direct role for Shp1 in negatively regulating signaling

from the TCR. Developing thymocytes undergo a stringent process of negative and selection in

which the strength of TCR signaling dictates survival and death. Although the thymus of

motheaten viable mice contains normal proportions of thymocytes, various studies demonstrated

a role for Shp1 through the use of transgenic TCR receptors. These transgenic receptors fixed the

affinity of the TCR for thymus-presented antigens, allowing investigators to examine what role

Shp1 may have during positive and negative selection. Observations of increased negative

selection suggested that Shp1 was directly controlling T cell selection by negatively regulating

TCR signal strength in developing thymocytes [279-281]. Studies additionally suggested an

analogous role for Shp1 in limiting TCR signaling in mature T cells [282, 283]. A multitude of

studies have suggested TCR proximal targets of the phosphatase activity of Shp1 including CD3ζ

[284], Lck [285, 286], Fyn [285], Zap70 [283, 284], and Slp76 [287]. However, there remains no

consensus on the physiological Shp1 target within the TCR proximal signaling complex [288].

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There have also been suggestions that various inhibitory receptors expressed by T cells

mediate their effects through activation of Shp1. As previously discussed, the Ig superfamily

molecule PD-1 can recruit Shp1 to its ITIM and ITSM domains [260]. However, further

experiments suggested that Shp1 does not contribute to PD-1 function [262]. Another molecule

of the Ig superfamily, BTLA, has also been implicated in the inhibition of T cell activation [289].

Two ITIM motifs are found on the cytoplasmic tail of BTLA which mediate the recruitment of

Shp1 [290]. However, mutation of key tyrosine residues which mediate the recruitment of Shp1

to BTLA had no apparent impact on the function of BTLA [291].

Interleukin-10 (IL-10) is an anti-inflammatory cytokine that maintains immune tolerance

in part through its ability to damped effector T cell activation [292]. The dominant negative

C453S Shp1 mutant was able to block to the ability of IL-10 to suppress T cell proliferation,

suggesting that Shp1 may mediate the effects of IL-10 in T cells. Additionally, the effects of

another anti-inflammatory cytokine, transforming growth factor β (TGFβ), were suggested to be

Shp1-dependent in experiments demonstrating that T cells from motheaten mice are resistant to

TGFβ-induced suppression [293].

Surface expression of Ceacam1 is induced on activated T cells where it can provide

inhibitory signals by virtue of the two ITIM motifs, which bind to Shp1 and Shp2, contained

within its cytoplasmic domain [294-296]. Interestingly, Neisseria gonorrhoeae is known to

inhibit T cell responses through interactions with Ceacam1, which induces the recruitment and

activation of Shp1 [294]. T-cell immunoglobulin domain and mucin domain-3 (TIM-3) is a

potent negative regulator of T cell activation that has been demonstrated to play important roles

during chronic viral infections and cancer [297]. A recent report demonstrated that Ceacam1

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forms heterodimers with TIM-3 and is required for its inhibitory function. These data suggest

that Shp1 could play a role in Tim3-mediated T cell suppression [298].

In summary, numerous inhibitory molecules have been suggested to act through Shp1-

mediated suppression. For many, Shp1 binding to ITIM or ITSM motifs has been demonstrated.

However, the functional consequences of Shp1 recruitment and activation are much less clear,

leaving the importance of Shp1 in these inhibitory pathways uncertain.

Shp1 has also been suggested to regulate the differentiation of CD4+ T cells. Several

studies have reported an inhibition of Th1 cells [293, 299, 300] as well as Th2 cells [301]. The

differentiation of the various Th-lineages is thought to be primarily controlled by cytokines.

Many cytokines signal through activation of Janus kinases (Jaks) which phosphorylate

themselves as well as Signal transducer and activator of transcription (Stat) molecules. Upon

their phosphorylation and subsequent dimerization, Stat molecules translocate into the nucleus

where they direct transcription. Notably, Shp1 has been suggested to dephosphorylate both Jak

[270, 302, 303] and Stat [304, 305] molecules following cytokine signaling. Therefore, it could

be through the regulation of phosphorylation events downstream of cytokine signaling that Shp1

is affecting Th1 and Th2 T cells. For example, Jak2, which is important for promoting Th1 cell

differentiation and function, may be dephosphorylated by Shp1 [302]. This potentially explains

the reported enhanced Th1 cell function in the absence of Shp1.

Finally, Shp1 has been implicated in the regulation of an assortment of other signaling

pathways pertinent to T cell biology. Chemokine responsiveness may be controlled by Shp1.

Motheaten mouse-derived cell show hypersensitivity to CXCL12 stimulation [306].

Furthermore, ligation of CCR5 induces phosphorylation of the C-terminal tyrosines of Shp1

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[307]. Apoptosis may also be regulated by Shp1, as it has been suggested to inhibit Fas signaling

[308, 309], although subsequent work contradicts these findings [310].

The severe inflammatory phenotype of the natural Shp1 mutants have made it

challenging to study the role of Shp1 during in vivo T cell responses. The development of a

conditional Shp1 knockout mouse has circumvented this issue. The first report of conditional

deletion of Shp1 in T cells examined responses to infection with lymphocytic choriomeningitis

virus (LCMV) [311]. Investigators found a greater expansion of short-lived effector CD8+ T

cells in the absence of Shp1. By contrast, no impact was found for the expansion or survival of

long-term memory T cells. A subsequent study examined the consequences of conditional Shp1-

deficiency on anti-cancer immunity [312]. TCR transgenic T cells were injected into mice

bearing a leukemia expressing a cognate antigen. Shp1-deficient T cells induced superior

anti-leukemia immunity and greater survival than wild type T cells. These experiments

demonstrate that Shp1 has an important role in regulating T cell biology in vivo.

Going forward, these conditional knockout mice will be a great boon to our

understanding of the role of Shp1 plays T cell biology. While many studies have linked Shp1

function with antagonism of signaling downstream of the TCR and the various other receptors

outlined above, they were largely performed with cells from homozygous motheaten or

motheaten viable mice. These mice are systemically inflamed, a factor which may have impacted

studies examining Shp1 function in T cells. Therefore, the use of conditional T cell knockout

mice, which lack the severe inflammation of motheaten mice [311], will provide an unobscured

view into the role of Shp1.

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Thesis outline and goals

The fate of an immune cell is governed by a series of molecular checks and balances:

phosphorylation and dephosphorylation, ubiquitination and deubiquitination, synthesis and

degradation, export and import. The loss of one of these regulatory balances can dramatically

alter immune cell function. In particular, some molecules are known to limit cell activation and

may therefore be broadly classified as negative regulatory molecules. This thesis will examine

the function of several of these molecules in regulating T cell and DC biology. Specifically, our

studies will focus on the roles of these molecules both during immune cell activation as well as

quiescence.

The phosphatase Shp1 is a potent negative regulator of immunity, as displayed by the

inflammatory phenotype of motheaten mice. More specifically, negatively regulatory functions

for Shp1 in T cell activation have been described by numerous reports. However, previous

studies may have been confounded by the systemic inflammation of motheaten mice.

Chapter III aims to determine the cell-intrinsic role of Shp1 in regulating fundamental aspects

of T cell biology. Towards this aim, we generated conditional T cell knockout mice and

investigated what impact the loss of Shp1 had on T cell development, homeostasis, activation,

and differentiation.

Previous work from our laboratory identified Nfkb1 as a negative regulator of DC

function. Specifically, without Nfkb1, DCs are able to induce tissue-specific immune pathology

without the requirement for exogenous stimulation. As Nfkb1encodes for both the IκB-like p105

protein and the NFκB subunit p50, it remained unclear which protein was contributing the

negative regulation of functional DC maturation. Chapter IV aims to address this issue through

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the use of two mouse strains, the Nfkb1 knockout which lacks both p50 and p105, and the p105

knockout which retains expression of p50. With these mice, we endeavored to determine distinct

functions of each protein in the regulation of DC maturation and function.

A20 is a key negatively regulator of inflammation and immunity which regulates the

NFκB signaling axis through the modulation of ubiquitination. Previous conditional knockout

studies have identified a critical role for A20 in inhibiting activation of DCs. Chapter V aims to

confirm this finding using A20-deficient DCs and our laboratory’s model of DC-induced

immune pathology. Through comparison of A20- and Nfkb1-deficient DCs, we investigated the

possibility that a core molecular phenotype may be associated with spontaneous DC maturation.

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Chapter II

Materials and Methods

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Mice

Shp1 floxed (Shp1fl/fl

) [274], A20 floxed (A20fl/fl

)[182], p105 knockout (p105-/-

)[313], P14 [314]

and CD4cre [315] mice have been described. OTI, CD45.1, Thy1.1, IL-4 knockout (IL-4-/-

),

Nfkb1 knockout (Nfkb1-/-

), and CD11c-cre mice were obtained from the Jackson Laboratory.

Shp1 and A20 floxed mice were generated and maintained on the C57Bl/6 background. P105

mice were generated with CJ7 embryonic cells. These mice were obtained after five backcrosses

with C57Bl/6 and were backcrossed an additional five times. Experiments with Shp1 and A20

conditional knockout mice used Cre-negative littermates as wildtype controls. C57Bl/6 mice

from the Jackson laboratory were used as controls for experiments with Nfkb1-/-

and p105-/-

mice.

Mice were housed in the Ontario Cancer Institute animal facility in accordance with institutional

regulations. Animal protocols were approved by the Ontario Cancer Institute Animal Care

Committee.

Bone marrow chimeras were generated by i.v. injection of a total of 5x106 bone marrow cells

into irradiated mice. Recipient mice received 900cGy of radiation delivered by an X-RAD320

(PXi) two hours before reconstitution. Chimeras were analyzed 3 months (Chapter III) or 6

months (Chapter IV) after their generation.

Western Blots and EMSA

Indicated cell populations were lysed in NP-40 lysis buffer (Roche) (Chapter III) or RIPA

buffer (Chapters IV, V). Equal amounts of protein were resolved on NuPAGE 4-12% Bis Tris

gels (Invitrogen) and transferred onto PVDF membranes using an iBlot (Invitrogen). Membranes

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were blocked with 5% milk in TBS containing Tween-20 (Sigma), stained with antibodies

against Shp1 (Upstate), p105/p50 (Abcam), Rela, Relb, cRel, IκBα, β tubulin (Santa Cruz), and

actin (Sigma) and subsequently developed using ECL Plus (GE Healthcare).

Cytoplasmic and nuclear fractions were isolated from DCs using NE-PER kit (Thermo). The

isolated fractions were then assayed by EMSA using labeled AP-1 and NFκB consensus

oligonucleotides (Li-Cor) and visualized using a LiCoR Odyssey.

Flow cytometry and Cell sorting

For analytical flow cytometry of T cell and DCs, cells were stained with antibodies against CD5,

CD8, CD25, CD45.2, CD40,CD69, CD70, Thy1.1, Thy1.2, TNFα, Vα2, pStat6 (BD), CCR7,

CD4, CD24, CD44, CD62L, CD80, CD86, CD127, Granzyme B, OX40L, IFNγ, IL-2, IL-4, and

IL-5, MHCI, MHCII, (eBioscience). Tetramer staining was performed on blood or splenic

lymphocytes using tetramers prepared from H-Db:KAVYNFATM (gp33) and H-

2Kb:AVYNFATC (gp34) (NIH) monomers and fluorophore-conjugated extravidin (Invitrogen).

Intracellular cytokine staining was performed using Cytofix/Cytoperm and Perm Wash buffers

(BD). Data were collected on a FACSCalibur or FACSCanto (BD)(Chapter III) and

FACSCanto II or Fortessa (Chapters IV, V) and analyzed with FlowJo software (TreeStar).

T cells were isolated using negative magnetic selection kits: Pan T Isolation Kit II (Miltenyi

Biotec) (Chapter III), CD8 T cell Isolation Kit (Miltenyi Biotec) (P14 T cells, Chapters IV, V)

and CD4 T cell Isolation Kit (Miltenyi Biotec)(Smarta T cells, Chapter IV). For FACS, splenic

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T cells were stained with α-CD44 (eBioscience), and thymocytes were stained with α-CD4

(eBioscience) and α-CD8 (BD). Cell sorting was performed on a MoFlo (Beckman Coulter), and

the purities of target populations were routinely >90%.

In vitro T cell assays

Cells were cultured in RPMI-1640 supplemented with 10% FBS, L-glutamine,

β-mercaptoethanol, penicillin and streptomycin. For thymocyte assays, P14 CD8+ single positive

thymocytes were co-cultured in round-bottom 96-well plates at a 1:10 ratio with irradiated

splenocytes from C57BL/6 mice (Chapter III). Thymocyte proliferation was induced by

stimulation with gp33 (KAVYNFATC). T cell stimulations were performed in flat-bottom 96-

well plates containing α-CD3 and α-CD28 (eBioscience), cross-linked with 10µg/mL α-hamster

IgG (Jackson ImmunoResearch) (Chapter III). For proliferation assays, cells were stained with

2.5µM CFSE (Invitrogen) prior to culture. Cytokine production assays were performed by

adding GolgiPlug (BD) to cultures one hour after stimulation, and then harvesting cells for

staining 5 hours later. CD4+ T cell stimulation assays were performed by culturing naïve CD4+ T

cells with α-CD3 and α-CD28 for three days, as above, with the addition of 50 U/mL IL-2

(eBioscience) for 3 more days. Cells were then re-stimulated with PMA and ionomycin

(eBioscience). To assess Stat 6 activation, cells were stimulated with IL-4 (eBioscience) in 96-

well round-bottom plates under the indicated conditions, before fixation with Lyse/Fix Buffer

(BD), permeabilization with PermBuffer III (BD), and analysis of pStat6 by flow cytometry.

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BMDC Generation

Bone marrow cells were flushed from the femur and tibia and then cultured in RPMI-1640

supplemented with 40ng/mL GM-CSF (Peprotech), 10% LPS-free FBS (HyClone), L-glutamine,

β-mercaptoethanol, penicillin and streptomycin. Cells were seeded into 6-well non-tissue culture-

treated plates. Cultures were maintained through the addition of additional GM-CSF-

supplemented media on day 3 and half-media changes on days 6 and 8. Cells were harvested for

experimentation between days 7 and 10.

Co-culture Assays

In preparation for co-culture (Chapters IV, V), BMDCs were stimulated, or not, with 10 M

CpG ODN 1826 (IDT) overnight, followed by the addition of gp33 peptide

(KAVYNFATC)(Washington Biotechnology) or AV peptide (SGPSNTPPEI) (Washington

Biotechnology) for 3 further hours of incubation. BMDCs were then harvested, washed, and

plated at 1.0x104 cells per well. Splenic P14 or Smarta T cells were isolated using magnetic

sorting, fluorescently labeled using e450 Cell Proliferation Dye (eBiosciences), and plated at

1.0x104

cells per well. Cells were cultured in RPMI-1640 supplemented with 10% LPS-free FBS

(HyClone), L-glutamine, β-mercaptoethanol, penicillin and streptomycin in flat-bottom 96-well

plates for up to 3 days. Some wells were supplemented with a neutralizing TNFα antibody

(eBiosciences). Transwell experiments were performed using 24-well 0.4µm membrane tissue

culture plates (Corning).

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In vivo Assays

For IL-4 neutralization experiments (Chapter III), 200µg of α-IL-4 IgG1 (11B11, BioXCell) or

an isotype control (HRPN, BioXCell) were administered to mice by i.p. injection. Mice were

sacrificed for analysis five days post-injection.

In preparation for vaccination of RIP-gp mice (Chapters IV, V), BMDCs were cultured

overnight with or without 10 M CpG ODN 1826 (IDT). The following day cultures were pulsed

for 3 hours with gp33(KAVYNFATC), gp276(SGVENPGGYCL), and

gp61(GLNGPDIYKGVYQFKSVEFD) peptides. 2x106 BMDCs were then intravenously infused

RIP-gp mice by tail vein injection. The onset of diabetes was monitored by measurement of

blood glucose using Accu-chek III Glucometers and Chemstrips (Roche).

ELISAs and Bead Arrays

Sera were isolated from 12-16 week-old littermate pairs using microtainer serum separator tubes

(BD), and duplicate samples were analyzed by ELISA for IgE (BioLegend) and IgG2a

(eBioscience), following the manufacturer’s instructions (Chapter III). Supernatants harvested

from DC cultures following their stimulation with CpG were assayed using the Mouse

Inflammation CBA kit (BD) (Chapters IV, V).

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Chapter III

Shp1 regulates T cell homeostasis by limiting IL-4 signals

Dylan Johnson, Lily Pao, Salim Dhanji, Kiichi Murakami, Pamela Ohashi, and Benjamin Neel

This chapter has been adapted from a publication:

Johnson DJ, Pao LI, Dhanji S, Murakami K, Ohashi PS, Neel BG. 2013. Shp1 regulates T cell

homeostasis by limiting IL-4 signals. J Exp Med 210: 1419-31

All authors contributed to the design of experiments.

DJ performed all experiments.

LP, SD, and KM performed supporting experiments not displayed here.

DJ, PO, and BN wrote the manuscript.

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Introduction

T cells are characterized by their ability to expand dramatically in an antigen-specific manner

during an immune challenge. Following an initial immune response, a small proportion of

responding T cells survive and give rise to memory cells [316]. Memory T cells express elevated

levels of CD44 and can be divided further into central-memory (CD62Lhi

CCR7hi

) and effector-

memory (CD62Llo

CCR7lo

) compartments. However, not all T cells that display the phenotype

of memory cells are the product of a classical antigen-specific immune response [317]. For

example, memory phenotype cells are found in unimmunized mice, including those raised in

germ-free and antigen-free conditions [318, 319]. The precise ontogeny of such cells remains

elusive, although several mechanisms by which naïve cells can adopt a memory-phenotype have

been characterized. Naïve T cells introduced into lymphopenic environments adopt a memory

phenotype through a process of homeostatic proliferation in response to IL-7 and MHC [320,

321]. Additionally, increased production of IL-4 has been linked to the development of memory-

phenotype “innate” T cell populations in studies of several knockout mouse models [322].

The T cell response is tightly regulated by the balance of phosphorylation and

dephosphorylation of intracellular signaling molecules. Shp1 (encoded by Ptpn6) is a protein-

tyrosine phosphatase (PTP) expressed ubiquitously in hematopoietic cells, and has been broadly

characterized as a negative regulator of immune cell activation [323, 324]. The physiological

relevance of Shp1 as a key negative regulator of the immune response is illustrated by the

motheaten (me) and motheaten-viable (mev) mutations, which ablate Shp1 expression or greatly

reduce Shp1 activity, respectively [265, 266]. Homozygous me/me or mev/me

v mice (hereafter,

referred to collectively as “me” mice) suffer from severe systemic inflammation and

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autoimmunity, which result in retarded growth, myeloid hyperplasia, hypergammaglobulinemia,

skin lesions, interstitial pneumonia, and premature death. More recently, a study has identified a

third allele of Ptpn6, named spin, which encodes a hypomorphic form of Shp1 [325]. Mice

homozygous for spin develop a milder auto-immune/inflammatory disease that is ablated in

germ-free conditions.

Shp1 has been implicated in signaling from many immune cell surface receptors [241,

326], including the TCR [283, 288], BCR [267, 327], NK-cell receptors [328, 329], chemokine

receptors [306], FAS [308-310], and integrins [273, 330]. Shp1 also has been demonstrated to

regulate signaling from multiple cytokine receptors by dephosphorylating various Jak [270, 302,

303] and/or Stat [304, 305] molecules. Several of these cytokines are pertinent to T cell biology.

For example, Stat 5 is an essential mediator of signals from IL-2 and IL-7 [331]. IL-4 signaling

results in Stat 6 phosphorylation and has potent Th2 skewing effects. Additionally, IL-4 has

mitogenic effects on CD8+ T cells [331]. Notably, mutation of the ITIM in IL-4α results in

ablation of Shp1 binding and hypersensitivity to IL-4 stimulation [305], implicating Shp1 as a

regulator of this cytokine receptor.

Although development of the me phenotype does not require T cells [332, 333], several

aspects of T cell biology reportedly are controlled by Shp1 [288]. Most previous studies that

examined the role of Shp1 in T cells used cells derived from me/me or mev/me

v mice [279, 280,

334, 335], or cells expressing a “dominant negative” allele of Shp1 [281, 283, 335]. Several

such reports have concluded that Shp1 negatively regulates the strength of TCR signaling during

thymocyte development and/or peripheral activation [279-281, 334, 335]. Despite the large

number of studies that implicate Shp1 in control of TCR signaling, there is no consensus on

which component of the TCR signaling cascade is targeted by the catalytic activity of Shp1.

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Suggested Shp1 targets downstream of T cell activation include TCRζ [284], Lck [285, 286],

Fyn [285], ZAP-70 [283, 284], and SLP-76 [287]. Shp1 also is implicated in signal transduction

downstream of several immune inhibitory receptors that negatively regulate T cell activity, such

as PD-1 [260], IL-10R [336], CEACAM1 [337], and CD5 [338].

The severe inflammation characteristic of the me phenotype might have confounded

studies examining the cell-intrinsic role of Shp1 in various hematopoietic cell types. We

previously generated a floxed Shp1 allele that facilitates analysis of the role of Shp1 in various

lineages [274]. Previous studies have used this approach to study the role of Shp1 in T cells

during anti-viral and anti-tumour immune responses, respectively [311, 312]. However, a more

fundamental analysis of the cell-intrinsic role of Shp1 during T cell development, homeostasis

and activation has not been reported. Here we provide evidence that a major role for Shp1 in T

cells is to maintain normal T cell homeostasis through negative regulation of IL-4 signaling.

Results

T cell-specific deletion of Shp1

To examine the cell-intrinsic role of Shp1 in T cells, we generated mice homozygous for a floxed

allele of Ptpn6 that also expressed CD4-cre. As expected, absence of Shp1 expression was

detected in double positive (DP) thymocytes and their progeny (Figure III-1A). Shp1fl/fl

CD4-cre

mice did not develop overt autoimmunity or inflammation (Figure III-1B), consistent with prior

work showing that the motheaten phenotype is T cell-independent [332].

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Figure III-1. T cell specific deletion of Shp1. A. The indicated thymocyte populations were

FACS sorted and Shp1 expression was determined by immunoblot. B. Frozen sections of organs

from Shp1fl/fl

and Shp1fl/fl

CD4-cre littermates were stained with hematoxylin and eosin. Images

are representative of 4 littermate pairs. Scale bars indicate 250µm.

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Thymocytes develop normally in the absence of Shp1

Next, we asked if Shp1 plays a role in thymocyte development. Shp1 conditional knockout mice

contained normal proportions of double negative (DN1->DN4), DP, and CD4+ and CD8+ single

positive (SP) thymocyte populations (Figure III-2A). There also was no change in the absolute

number of cells contained within each developmental compartment of the thymus (Figure III-

2B), nor did we detect any differences in the staining of CD4+ or CD8

+ SP thymocytes for the

markers CD5, CD69 or CD24 (Figure III-2C). Together, these data suggest that Shp1 is

dispensable for thymocyte development.

Previous studies of mice expressing TCR transgenes identified a role for Shp1 in

thymocyte selection [279-281, 335]. We crossed our T cell-specific Shp1 conditional knockout

mice with mice expressing the P14 TCR transgene, an MHC-I restricted TCR composed of Vα2

and Vβ8.1, which recognizes the gp33-41 epitope of lymphocytic choriomeningitis virus

(LCMV) in the context of H-2Db

[314]. Surprisingly, Shp1-deficient P14 transgenic mice had

normal proportions and numbers of thymocyte subsets, including the positively selected CD8+

SP population (Figures III-2D,E). The CD8+ SP population also expressed similar levels of

CD5, CD69, and Vα2, suggesting that positive selection was unaltered by the absence of Shp1

(Figure III-2F). Shp1 deficiency also did not influence the selection of the OT-I or OT-II TCR

transgenes (data not shown). To verify that Shp1 does not regulate thymocyte selection, we

performed in vitro thymocyte stimulations. Wild type and Shp1-deficient P14 thymocytes

required an equivalent concentration of gp33 to induce proliferation. Furthermore, Shp1 had no

impact on the extent of proliferation induced by gp33 (Figure III-2G). Together, these results

suggest that Shp1 is not essential for regulating thymocyte selection or TCR signaling threshold.

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Figure III-2. Thymocytes develop normally in the absence of Shp1. A. Thymocytes from

Shp1fl/fl

and Shp1fl/fl

CD4-cre mice were stained with antibodies specific for various surface

markers. CD8 and CD4 profiles for total thymocytes and CD44 and CD25 expression for gated

DN thymocytes are shown in A. Total cell numbers for each population are shown in B; n = 4.

C. Mature CD4+ and CD8

+ SP thymocytes were evaluated by flow cytometry for the expression

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of the surface markers CD24, CD69 and CD5. Thymocytes from P14 Shp1fl/fl

and P14 Shp1fl/fl

CD4-cre mice were evaluated using a similar panel of antibodies. D. Thymocytes were stained

with α-CD4 and α-CD8 antibodies, and absolute number of the various subsets are shown in E,

n=3. F. CD8SP P14 thymocytes were gated and CD24, CD69, CD5, and Vα2 expression was

assessed by flow cytometry. G. CD8 SP P14 thymocytes stained with CFSE were cultured for

two days with irradiated splenocytes and the indicated concentration of gp33. Statistical analyses

were performed by one-way ANOVA (B) or Student’s t-test (E), ns p≥0.05. Values for B and E

are displayed as ± standard error.

Memory-phenotype T cells accumulate in Shp1 conditional knockout mice

Next, we examined the phenotype of peripheral T cells from Shp1fl/fl

CD4-Cre mice compared

with control Shp1fl/fl

mice. There was no difference in the total number of T cells contained

within wildtype and mutant mice (data not shown). However, we found that spleens from

Shp1fl/fl

CD4-cre mice were enriched for T cells expressing elevated levels of CD44 (Figure III-

3A, B). This increase was observed in the CD4+ and the CD8

+ T cell compartments, although

the increase in the CD8+ CD44hi

population was more prominent. Shp1-deficient lymph node

and blood T cells also displayed a CD44hi

phenotype (data not shown). By contrast, CD44 levels

were normal in mature SP-thymocytes, indicating that Shp1-deficient T cells become CD44hi

following thymic egress (Figure III-3A). A previous study of me mice implicated Shp1 in

control of regulatory T cell (Treg) development [339]. However, we found no difference in the

frequency of Tregs in T cell-conditional Shp1 knockout mice (data not shown).

Elevated CD44 expression is associated with activated and memory T cells. The CD44hi

population in our mice appeared to have a memory phenotype, as they did not express the

activation markers CD25 or CD69 (data not shown). Memory T cells can be divided further into

central memory (CD44hi

CD62Lhi

CD127hi

CCR7hi

) and effector memory (CD44hi

CD62Llo

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75

CD127lo

CCR7lo

) populations. The CD4+ CD44

hi T cell populations from wild type and T cell

conditional Shp1 knockout mice were composed of a mixture of CD62Lhi

and CD62Llo

cells

(Figure III-3C). The CD4+ CD44

hi populations also displayed heterogeneous expression of

CD127 and CCR7. Together, these findings suggest that the CD4+ memory-phenotype

compartment contained a mixture of effector- and memory-phenotype cells. By contrast, the

CD8+ CD44

hi populations contained a high proportion of CD62L

hi CD127

hi CCR7

hi cells,

suggesting a prominent central-memory phenotype.

The expression of these markers was identical in Shp1-deficient memory-phenotype cells

and the naturally occurring memory-phenotype population present in wild type mice. This

finding suggests that the Shp1-deficient CD44hi

population reflects an expansion of normally

occurring memory-phenotype T cells. Notably, the central memory phenotype of the CD44hi

populations is consistent with the phenotype of naïve T cells that have undergone homeostatic

expansion [317]. To test if the memory-phenotype population was a consequence of an antigen-

specific T cell expansion we examined the expression of CD44 on P14 TCR transgenic T cells.

However, CD44 expression also was elevated in Shp1-deficient P14 T cells, demonstrating that

the accumulation of memory-phenotype cells is not driven by a response to (a) specific

endogenous antigen(s) (Figure III-3D).

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Figure III-3. Shp1 restricts the development of memory-phenotype T cells. T cells from

Shp1fl/fl

or Shp1fl/fl

CD4-cre mice were stained with monoclonal antibodies against the indicated

cell surface molecules A. Mature SP thymocytes and splenic T cells were assayed for CD44

expression by flow cytometry. B. Percentage of splenic T cells with CD44hi

phenotype,

displayed as ± standard error; n=8. C. Splenic T cells, gated based on expression of CD4, CD8

and CD44, were stained with antibodies against the indicated markers. D. Expression of CD44

on splenic T cells from P14+ mice. Cells are gated on the CD8+ Vα2+ population. Statistical

analysis of data in B was performed by Student’s t-test, ** p<0.01.

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T cells respond normally to TCR stimulation in the absence of Shp1

To test whether Shp1 plays a role in regulating TCR sensitivity, T cells from Shp1fl/fl and

Shp1fl/fl

CD4-cre mice were stimulated in vitro with α-CD3 ± α-CD28. Total CD4+ T cells from

Shp1 conditional knockout animals displayed increased proliferation, compared with wild type

CD4+ T cells, when stimulated with α-CD3 or α-CD3 + α-CD28. (Figure III-4A). Shp1-deficient

CD8+ T cells stimulated with α-CD3 displayed dramatic hyperproliferation, although this

difference was eliminated upon addition of α-CD28. To determine if the enriched memory-like

population in Shp1 conditional knockout animals was responsible for the enhanced proliferation,

T cells were sorted for CD44, and the CD44lo

population was then stimulated with α-CD3 ±

α-CD28 (Figure III-4B). Notably, the enhanced proliferation of Shp1-deficient (total) T cells was

eliminated when the large memory-phenotype population was removed. This result indicates that

the apparent hyperproliferative response of Shp1-deficient T cells reflects the intrinsically more

robust response of memory-phenotype cells, rather than the effects of Shp1 on TCR

responsiveness per se.

We also examined the capacity of Shp1-deficient T cells to produce cytokines in response

to TCR stimulation. When cells were gated based on their CD44 expression, there was no

difference in the ability of wild type or Shp1-knockout T cells to produce IL-2 (Figure III-4C) or

TNFα (Figure III-4D) in response to stimulation with α-CD3 and α-CD28. In contrast to previous

studies [280, 311], these data suggest that Shp1 does not have a role in regulating TCR

sensitivity in peripheral T cells.

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Figure III-4. Shp1-deficient T cells exhibit normal responses to TCR stimulation. A. T cells

were isolated from spleens, labeled with CFSE, and cultured on a 96-well plate coated with

cross-linked antibodies against CD3± CD28. Cells were harvested 3 days later, stained for CD4

and CD8, and analyzed by flow cytometry. B. Splenic T cells were sorted into CD44hi

and

CD44lo

populations by FACS, labeled with CFSE and cultured on a 96-well plate coated with

cross-linked antibodies against CD3 and CD28 at the indicated concentrations. Cells were

analyzed as in A. C, D. Cells were treated as in A, harvested six hours post-stimulation and

analyzed for IL-2 (C) and TNFα (D) expression by intracellular staining; n=2. Data are

representative of three independent experiments. Values for C,D are displayed as ± standard

error.

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T cells skew to Th2 in the absence of Shp1

We next investigated if Shp1 has a role in controlling the differentiation of CD4+ Th cells. Naïve

CD4+ T cells were stimulated in vitro with α-CD3 and α-CD28, followed by three days of

culture in the presence of IL-2. Upon re-stimulation, a significantly lower proportion of Shp1-

deficient T cells expressed IFNγ compared with controls (Figure III-5A,B). Additionally, there

was a significant increase in the frequency of IL-4-producing cells. By contrast, Shp1-deficient

T cells stimulated immediately following their isolation exhibited normal production of IL-4

(data not shown), suggesting that Shp1-deficient T cells are not pre-programmed for IL-4

production. Together, these findings suggest that Shp1 negatively regulates Th2 differentiation in

vitro. To examine whether the in vitro Th2 bias is also seen in vivo, we examined serum antibody

levels. Indeed, the serum concentration of IgE, the prototypic Th2 antibody isotype, was

approximately 50-fold higher in knockout mice compared with controls (Figure III-5C). By

contrast, knockout and wild-type mice had similar levels of serum IgG2a, a Th1-driven isotype.

Therefore, Shp1 regulates Th2 skewing in vitro and in vivo.

Th2 differentiation is regulated by a transcriptional network that includes IL-4R-induced,

pStat6-directed transactivation of the master Th2 transcription factor GATA-3 [340]. To

determine if Shp1 regulates signaling downstream of IL-4R in T cells, we stimulated wild type

and Shp1-deficient T cells with IL-4, and measured Stat 6 tyrosyl phosphorylation. Shp1

deficiency did not affect the dose-response curve for IL4-evoked Stat 6 phosphorylation (Figure

III-5D). To measure the kinetics of pStat6 dephosphorylation, T cells were pulsed with IL-4 for

30 minutes, followed by three washes to remove residual cytokine. Wild-type T cells showed

robust Stat6 tyrosyl phosphorylation upon IL4 stimulation, followed by a loss of the pStat6

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signal two hours later. By contrast, Shp1-deficient T cells maintained high levels of Stat6

phosphorylation at two and three hours post-cytokine withdrawal (Figure III-5E), indicating that

Shp1 is required for the efficient dephosphorylation of Stat6 following IL-4 stimulation.

Figure III-5. T cells skew to Th2 in the absence of Shp1. A. Naïve (CD44lo

) CD4+ T cells

were isolated, stimulated for three days with α-CD3/CD28, and then re-stimulated with

PMA/Ionomycin four days later. Cells were harvested and stained 6 hours post re-stimulation.

B. Cells were stimulated and analyzed as in (A). Percentages of cells staining positive for IFN γ

or IL-4. Data are representative of three independent experiments and are displayed as ± standard

error; n=4. C. Concentration of IgE and IgG2a in sera from the indicated unmanipulated

age-matched mice, horizontal bars represent sample means; n=8. D. T cells were stimulated with

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81

the indicated concentrations of IL-4 for 30 minutes and then fixed and stained for p-Stat6.

Histograms are gated on CD44lo

T cells. E. T cells were stimulated with IL-4 (10 ng/mL) for 30

minutes and were then washed three times with media. Cells were harvested at the indicated

times and analyzed as in D. Data are representative of three independent experiments. Statistical

analyses of data in B and C were performed by Student’s t-test, ns p≥0.05, * p<0.05, ** p<0.01.

Memory-phenotype cells in Shp1 conditional knockout mice are dependent on IL-4

We sought to determine if cell-intrinsic or –extrinsic forces were driving the formation of

memory-phenotype T-cells within Shp1 conditional knockout mice. Towards this aim, we

generated mixed bone marrow chimeras. Irradiated, congenically marked (CD45.1) hosts were

reconstituted with bone marrow cells from wild type mice (Thy1.1), conditional knockout mice

(Shp1fl/fl

CD4cre), or a 1:1 mixture of the two. In mice reconstituted with conditional knockout

bone marrow, there was a significant enrichment for both CD4+ and CD8+ memory-phenotype

T cells in comparison to mice reconstituted with wild type bone marrow (Figure III-6A,B).

Mixed bone marrow chimeras contained wild type T cells with a predominantly naïve phenotype

and knockout T cells with an enriched memory-phenotype population. This finding indicates that

Shp1 knockout T cell phenotype is cell intrinsic and not a response to altered cell-extrinsic

factors.

Previous studies have linked IL-4 to the abnormal expansion of memory-phenotype CD8+

T cells [322]. To test if the enhanced sensitivity of Shp1-deficient T cells to IL-4 was driving the

accumulation of memory-phenotype cells, we crossed our Shp1 conditional knockout mice to IL-

4-/- mice. The absence of IL-4 had no effect on the frequency of peripheral blood CD44hi

cells in

mice expressing Shp1 (Figure III-6C,D). However, lowering (IL4+/-

) or eliminating (IL4-/-

) IL-4

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82

expression in Shp1 conditional knockout mice resulted in a reduced percentage of CD4+ and

CD8+ CD44

hi cells in blood (Figure III-6D). The percentage of CD4

+ CD44

hi T cells returned to

wild type levels in double knockout mice, suggesting that IL-4 is essential for the development

of excess CD4+ CD44

hi T cells caused by the absence of Shp1. Decreasing IL-4 levels also

lowered the proportion of CD8+ CD44

hi T cells, although the percentage of these cells

consistently remained above wild type levels in all organs, suggesting that additional factors

contribute to CD8+ memory-phenotype T cell development in the absence of Shp1. Serum IgE

was undetectable in double knockout mice (data not shown), indicating that IL-4 is critical for

the elevated IgE levels detected in Shp1 conditional knockout mice.

To ask whether IL-4 also is required to maintain the increased memory-phenotype

population, mice were treated with a neutralizing α-IL-4 antibody [341]. Administration of α-IL-

4 to Shp1 conditional knockout mice resulted in a significant reduction in the levels of

CD4+CD44

hi T cells, which reached wildtype levels, and CD8

+CD44

hi T cells, although this

population remained elevated compared with controls (Figure III-6E,F). These data further

suggest that the enriched CD4+ memory-phenotype population is completely IL-4 dependent,

while other factors contribute to the expanded CD8+ population. In sum, IL-4 is required for both

the development and maintenance of the enriched population of memory-phenotype T cells

found within mice with Shp1-deficient T cells.

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Figure III-6. IL-4 is required for the accumulation of CD44hi

T cells in Shp1 conditional

knockout mice. A. Bone marrow from Thy1.1 and/or Shp1fl/fl

CD4-cre mice was transferred into

irratiated CD45.1 host animals in order to generate mixed bone marrow chimeras. CD44

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84

expression on splenic T cells was analyzed by flow cytometry. Flow plots are gated on

CD45.2+Thy1.1

+Thy1.2

- (Thy1.1) or CD45.2

+Thy1.1

-Thy1.2

+(Shp1

fl/fl CD4-cre) populations, as

well as CD4+ or CD8

+. B. Percentage of splenic T cells with a CD44

hi phenotype in bone marrow

chimeras from A.; n=6-7. C. CD44 expression on T cells in the blood of mice of the indicated

genotypes. D. Percentage of T cells with a CD44hi

phenotype in the indicated tissues of IL-4 and

Shp1-deficient mice; n=6-7. Brachial LN (BLN), cervical LN (CLN), inguinal LN (ILN) E.

CD44 expression of T cells in blood of mice given 200µg of α-IL-4 or an isotype control five

days before sacrifice. F. Percentage of T cells with a CD44hi

phenotype in the blood of mice

treated as in E. Data are representative of two independent experiments; n=4. Statistical analyses

of data in B, D, and F were performed by two-way ANOVA and Bonferroni post-test analysis,

ns p≥0.05, * p<0.05, ** p<0.01. Horizontal bars for B,D and F represent sample means.

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Discussion

The severe and complex phenotype of Shp1 mutant mice has hindered attempts at determining

the cell-autonomous role of Shp1 in various hematopoietic cell types. Floxed Shp1 mice provide

the best tool available for analyzing the cell-intrinsic consequences of Shp1 deficiency.

Therefore, we generated and analyzed mice with Shp1-deficient T cells. These mice lack the

overt autoimmunity of me mice, confirming that the absence of Shp1 in T cells alone is

insufficient for the development autoimmunity [332, 333].

Absence of Shp1 in T cells does not phenocopy the T cell phenotype of me mice

Shp1 has been identified as a negative regulator of Treg development in me mice [339]. By

contrast, our results demonstrate that Treg development is normal in T cells specifically lacking

Shp1. Furthermore, in contrast to the results of studies of me/me and mev/me

v -derived T cells,

we found that Shp1 deficiency has no effect on sensitivity to TCR stimulation. Several groups

have reported enhanced positive selection of me thymocytes expressing TCR transgenes [279,

280, 335]. By contrast, we find no change in the selection of thymocytes expressing either MHCI

(P14, OTI)- or MHCII (OTII)- restricted TCR transgenes. The P14, OTI, and OTII transgenes

are all “strongly” selected TCRs; consequently, Shp1 deficiency might affect the selection of

TCRs with lower affinity [342-344]. However, previous data implicated Shp1 in regulating the

selection of the DO11.10 TCR [279], which is believed to receive a stronger selecting signal than

OTII [343]. Together, our findings and the previous reports suggest that the selection defect

found in me mice is not due to a cell-autonomous effect of Shp1 deficiency, but rather is a

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consequence of Shp1 deficiency in another cell type (e.g., thymic DCs) and/or the severe

systemic inflammatory signals in these mice.

Likewise, the reported TCR hypersensitivity of peripheral T cells from me mice [280]

also is likely to be the result of systemic inflammation and the enhanced activation state of the

cells. Mice expressing a putative “dominant negative” (phosphatase-inactive) Shp1 allele in T

cells also were reported to show enhanced TCR sensitivity for thymocyte selection [281, 335]

and activation [283]. These mice lack the severe systemic inflammation of me mice, yet still

display TCR hypersensitivity. The discrepancy between these results and our study might be

explained by the ability of the catalytically impaired Shp1 mutant to interfere with the binding of

other SH2 domain-containing negative regulators to proteins with immunoreceptor tyrosine-

based inhibitory motifs (ITIMs) and immunoreceptor tyrosine-based switch motifs (ITSMs)

([288]. Over-expression of “dominant negative” Shp1 not only might outcompete wild type Shp1

for binding to ITIM- and ITSM- containing proteins, but also could block the binding of Ship

and/or Shp2 to these motifs, thereby inhibiting their roles in antagonizing TCR signaling as well.

In Shp1 conditional knockout T cells, competition for ITIM and ITSM binding is, if anything,

decreased, allowing other factors to interact with these proteins. Regardless, our results establish

that Shp1 is not essential for the negative regulation of TCR signaling. This study underscores

the value in separating cell-extrinsic and –intrinsic effects of Shp1 activity.

Shp1 restricts the development of memory-phenotype T cells

Our findings identify Shp1 as an important regulator of Th2 differentiation and T cell

homeostasis. Memory-phenotype T cells are characterized by their surface memory phenotype

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87

and may be characterized as innate T cells due to their ability to rapidly produce cytokines upon

TCR stimulation. Such cells have been hypothesized to play an important role in the early stages

of immune responses. IL-4 has been demonstrated to be essential for the accumulation of

memory-phenotype T cells in several other knockout mice. For example, mice deficient for

inducible T cell kinase (Itk) have an accumulation of memory-phenotype T cells in the thymus

[344], and the development of these CD44hi

thymocytes subsequently was shown to be

dependent on the production of IL-4 by NKT cells [345]. Mice deficient for Krüppel-like factor

2 (KLF2) or inhibitor of DNA binding 3 (Id3) also develop prominent CD44hi

populations in the

thymus that are dependent on IL-4 [345-347]. Like these memory-like or innate T cells, the

Shp1-deficient memory population expresses high levels of CD44 and has an increased capacity

to quickly produce cytokines following TCR stimulation. However, the memory-like populations

in Itk-, KLF2-, and Id3-deficient mice all arise during thymocyte development, whereas the

CD44hi

population in Shp1 conditional knockouts is restricted to the periphery, suggesting that a

distinct developmental pathway is responsible. Additionally, mixed bone marrow chimera

experiments revealed that loss of KLF2 in the T-lineage results in the development of memory-

phenotype T cells of both wild type and cKO origin. The KLF2 phenotype therefore is due to

elevated extracellular IL-4. By contrast, we found that the development memory-phenotype cells

in Shp1 cKO mice is cell intrinsic; wild type bystander cells in mixed bone marrow chimeras

maintain normal homeostasis. T cell protein-tyrosine phosphatase (TCPTP) T cell-conditional

knockout mice also accumulate CD4+ CD44

hi and CD8

+ CD44

hi populations in the periphery

[348]. Unlike Shp1-deficient T cells, however, the T cells in TCPTP conditional knockout mice

predominantly have an activated/effector-memory phenotype, and these mice develop systemic

inflammation and autoimmunity, including lymphoid infiltrates in the liver and lungs, elevated

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88

anti-nuclear antibodies, and increased germinal center formation. T cells from Shp1 conditional

knockout mice had a mixed effector/central memory phenotype, did not infiltrate tertiary tissues,

and promoted normal levels of germinal centre formation (data not shown).

We identified IL-4 as a critical factor driving the development and survival of CD4+

CD44hi

cells in Shp1fl/fl

CD4-cre mice. Elimination of IL-4 resulted in wild type levels of CD4+

CD44hi

cells in all lymphoid organs examined. IL-4 also promoted the development and survival

of CD8+CD44

hi cells, as this population was reduced substantially upon elimination of IL-4. In

contrast to the normalization of the number of CD4+CD44

hi cells, we observed only a partial

reduction of CD8+

CD44hi

cells in the blood, spleen, and lymph nodes of Shp1fl/fl

CD4-cre IL4-/-

mice. This finding indicates that there are other factors promoting the accumulation of CD8+

CD44hi

cells. The identity of these factors and their relative contributions to memory-phenotype

T cell development in various lymphoid organs remain to be elucidated.

Shp1 negatively regulates Th2 skewing

Previous studies of me-derived cells had identified Shp1 as a negative regulator of Th1

[293, 299] and Th2 differentiation [301], but it remained unclear if Shp1 has a cell intrinsic role

in regulating CD4+ T cell differentiation. Our results show that Shp1 restricts the development of

Th2 (but not Th1) cells in a cell-autonomous manner. This finding is congruent with the

autoimmune lung disease to which me mice succumb, which is characterized by excessive type-2

inflammation and can be partially limited by the elimination of IL-4, IL-13, or Stat6 [349].

However, given that CD4+ T cells are non-essential for the me lung phenotype [332, 333],

additional sources of IL-4 and IL-13 must drive lung inflammation in these mice.

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89

A critical step in the differentiation of Th2 cells is the IL-4 mediated activation of Stat 6

(Zhou et al., 2003), and Shp1 has been reported to antagonize IL-4/Stat 6 signaling. The

IL-4Rα-chain contains an ITIM that can interact with Shp1, Shp2, and Ship, and mutation of the

ITIM results in a hyperproliferative response to IL-4 in a myeloid cell line [305]. Additionally,

B-cells harboring the ITIM mutation have impaired dephosphorylation of Stat 6 [350], and

various hematopoietic cells types from me mice show enhanced Stat 6 phosphorylation in

response to IL-4 or IL-13 [350, 351]. However, Shp1 deficiency has been reported to have no

impact on the IL-4 induced phosphorylation of Stat 6 in CD4+ and CD8

+ T cells [352]. Our

results are in direct contrast, and suggest a cell-intrinsic mechanism for the regulation of IL-4

signaling by Shp1. A likely explanation for this discrepancy is that the previous study only

examined the induction of Stat 6 phosphorylation, whereas we found that Shp1 primarily

controls Stat 6 dephosphorylation. Regulation of Stat 6 phosphorylation probably explains how

Shp1 regulates Th2 differentiation.

IL-4 reportedly has diverse effects on peripheral T cells beyond its role in Th2

polarization. For example, IL-4 promotes the survival of resting T cells [353]. Consistent with

Shp1 deficiency having a more dramatic effect on CD8+ homeostasis, IL-4 has been

demonstrated to have potent mitogenic effects on CD8+ T cells [354] and T cells expressing a

constitutively active form of Stat 6 primarily adopt an activated phenotype [355]. Yet whether

IL-4 has a positive or negative effect on CD8+ activation, cytotoxicity, and memory formation

remains controversial [356-359]. The IL-4 hypersensitivity of Shp1-deficient T cells likely

contributes to the development of memory-phenotype T cells. However, enhanced IL-4

production by Shp1-deficient T cells is detected only following prolonged stimulation (Figure

III-5A,B) and not directly ex vivo. This finding demonstrates that in Shp1 conditional knockout

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animals, IL-4 facilitates the accumulation of memory-phenotype cells, but does not prime IL-4

production. Additionally, our mixed bone marrow chimera experiments demonstrate that IL-4

from Shp1-deficient T cells alone is insufficient to alter the homeostasis of wild type T cells.

Together these data strongly suggest that Shp1-deficient T cells are reacting to homeostatic

levels of IL-4, and that cell-intrinsic hypersensitivity to IL-4 signals is crucial for the increase in

memory-phenotype T cells and Th2 differentiation in the absence of Shp1. In this context, our

findings highlight that IL-4, like other IL2-Rγc cytokines, can have powerful regulatory effects

on CD4+ and CD8

+ T cell homeostasis and differentiation.

Concluding remarks

By analyzing T cell-conditional Shp1 knockout mice, we have delineated the cell

autonomous role of Shp1 in T cell development, homeostasis, and activation. What remains to be

determined is the precise role Shp1 plays in regulating T cells during various immune responses.

We have demonstrated that, in contrast to reports using me mice, Shp1 deficiency in T cells does

not have a major impact on thymocyte development. Rather, we have established that Shp1 is an

important regulator of peripheral T cell homeostasis. Through regulation of IL-4 signals in T

cells, Shp1 limits Th2 differentiation, IgE production, and critically limits the development of

memory-phenotype T cells.

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Chapter IV

The NFκB subunit p50 limits the Immunogenicity of Dendritic Cells

Dylan Johnson1,2

, Wenxin Chen2, Celine Robert-Tissot

2,Carlos Garcia-Batres

2, Pamela Ohashi

1,2

1Department of Immunology, University of Toronto, Toronto, Canada

2Campbell Family Institute for Breast Cancer Research, Toronto, Canada

All authors contributed to the design of experiments.

DJ performed all experiments with assistance from WC, CR, and CG.

DJ and PO wrote the manuscript.

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Introduction

Dendritic cells (DCs) are central regulators of the immune system, controlling both the

maintenance of tolerance and the induction of immunity [360] [361]. As professional antigen-

presenting cells (APCs), DCs directly control the activation of both CD4+ and CD8+ T cells,

thereby limiting or inducing inflammatory T cell responses. Therefore, a fundamental question of

DC biology is how these cells are regulated to carry out such divergent functions.

Homeostatic DCs are thought to have an immature or tolerogenic phenotype [362]. DC

immunogenicity may be induced through a process of DC maturation which is generally the

result of ligation of a pattern recognition receptor (PRR). The resulting signaling leads to the up-

regulation of MHCII, co-stimulatory molecules such as CD80 and CD86, and pro-inflammatory

cytokines such as TNFα, events which directly enhance the ability of DCs to promote T cell

activation. While DC immaturity may be often considered a default state, mounting evidence

suggests that intrinsic factors are actively required to maintain immaturity [363]. While the

molecular events which occur during DC activation have been extensively described [364],

comparatively, our understanding of the molecular program that maintains DC immaturity is

limited.

In order to better understand the underpinnings of DC maturation, our laboratory has

developed a strategy that allows us to test the immunogenicity of DCs in vivo[365, 366]. This

model employs the RIP-gp mouse which expresses the lymphocytic choriomeningitis virus

(LCMV) glycoprotein (gp) in pancreatic islet beta cells under the control of the rat insulin

promoter (RIP). In the RIP-gp mouse, the generation of potent T cell immunity against LCMV-

gp results in pancreatic islet infiltration and CD8+ mediated destruction of LCMV-gp expressing

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beta cells, and consequently the induction of diabetes. Vaccination of RIP-gp mice with bone

marrow-derived dendritic cells (BMDCs) pulsed with LCMV-gp peptides provides a means to

assay the ability of DCs to induce T cell activation. Unstimulated wild type DCs fail to induce

diabetes in RIP-gp mice. By contrast, DCs stimulated with a TLR ligand such as CpG DNA or

LPS are able to activate an anti-gp T cell response and thereby lead to the induction of diabetes.

The NFκB family of transcription factors is well known for its role in leukocyte

activation downstream of PRRs and antigen receptors. However, there is a growing appreciation

of anti-inflammatory functions of NFκB proteins. In particular, the gene Nfkb1, which encodes

for both p105 and its proteolytic cleavage product p50, has been suggested to have both pro-

inflammatory and anti-inflammatory functions [367]. Nfkb1-deficient mice are resistant to

experimental autoimmune encephalitis [368] and collagen induced arthritis [369], confirming a

role for p50 and/or p105 in producing inflammatory responses. However, NFκB p50 has also

been suggested to limit inflammation. Studies have suggested that p50 homo-dimers may bind to

κB sites within the TNFα promoter thereby blocking its transcription [370-372]. Furthermore,

p105 has IκB-like activity and may therefore prevent the nuclear translocation of inflammatory

NFκB dimers [373].

Our laboratory has previously identified a role for Nfkb1 in regulating the maturation of

DCs. BMDCs from Nfkb1-deficient mice do not require stimulation with a TLR ligand in order

to induce diabetes following their transfer into RIP-gp mice [374]. This finding suggests that

Nfkb1 is part of a molecular program which maintains DCs in a non-immunogenic state. There

are several potential mechanisms by which loss of Nfkb1 may impact DC homeostasis. It may be

the loss of p50-containing homo- or hetero-dimers which is the key event driving the functional

maturation of DCs. Alternatively, the IκB-like functions of p105 may be crucial for maintaining

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the DCs in a non-immunogenic state. In order to delineate these two possibilities, we have

obtained p105-/-

mice which transcribe and translate p50 directly and thereby express p50 but not

p105 [375, 376]. Mice specifically lacking p105 suffer chronic inflammation, demonstrating the

role of p105 in restricting pro-inflammatory signaling. Furthermore, p105 has been shown to

regulate IL-12 production in macrophages [377], suggesting that p105 may have an important

role in APC function. By comparing DCs generated from Nfkb1-/-

and p105-/-

mice we aim to

define the specific roles of p50 and p105 in regulating DC maturation. Furthermore, this analysis

will lead to a greater understanding of the phenotype and molecular profile of functionally

mature DCs.

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Results

Generation of DCs lacking NFκB p50 and p105

In order to elucidate the roles of p50 and p105 in DC biology, DCs were generated from

the bone marrow cells of Nfkb1-/-

and p105-/-

mice by culturing them with granulocyte-

macrophage colony stimulating factor (GM-CSF) for 8-10 days. Both Nfkb1-/-

and p105-/-

cultures generated predominantly CD11bhi

CD11chi

DCs suggesting that neither p50 nor p105 is

required for the development of DCs (Figure IV-1A). We then confirmed the expression of

Nfkb1 proteins in our cultured DCs; Nfkb1-/-

DCs lacked expression of both p50 and p105 while

p105-/-

DCs lacked p105 while having slightly elevated expression of p50 in comparison to wild

type DCs (Figure IV-1B).

We went on to analyze the surface phenotype of Nfkb1-/-

and p105-/-

DCs by flow

cytometry (Figures IV-1C, -1D). Both Nfkb1-/-

and p105-/-

DCs had slightly elevated expression

of MHCI in comparison to wild type DCs before and after treatment with the TLR9 ligand CpG

ODN. Next, we found that Nfkb1-/-

DCs had slightly lower MHCII expression compared to wild

type DCs and were unable to upregulate MHCII in response to CpG. By contrast, p105-/-

DCs

have slightly elevated basal MHCII expression. Furthermore, they upregulated MHCII to a

greater extent than wild type DCs in response to CpG. A similar pattern emerged for co-

stimulatory molecules of the B7 family. We observed that p105-/-

DCs had higher basal

expression of both CD80 and CD86 than did wild type DCs. Moreover, upon stimulation with

CpG, p105-/-

DCs demonstrated enhanced up regulation of CD80 and CD86. Nfkb1-/-

DCs,

however, had low basal expression of CD80 and CD86 and were unable to upregulate CD80 and

CD86 in response to CpG.

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Figure IV-1. Generation of DCs lacking NFκB p50 and p105. A. Bone marrow cells from

C57BL/6, Nfkb1-/-

, and p105-/-

mice were cultured for 8-10 days in GM-CSF to generate

BMDCs. Expression of CD11b and CD11c was measured by flow cytometry. B. BMDCs were

lysed and the expression of NFκB p105 and p50 were determined by Western blot. C. BMDCs,

with or without overnight stimulation with CpG, were stained with monoclonal antibodies

against the indicated markers and analyzed by flow cytometry. D. Quantification of geometric

mean fluorescence of markers analyzed in C, n=3. E. Following overnight culture, supernatant

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from BMDCs were collected and assayed by bead array for the presence of the indicated

cytokines, n=3. Statistical analyses for D and E were performed by two-way ANOVA with

Tukey’s test. Differences within treatment groups are indicated, * p<0.05, ** p<0.01. Data are

representative of at least 3 independent experiments.

We additionally examined molecules of the tumour necrosis factor superfamily (TNFSF),

namely CD40, CD70, and OX40L. DCs lacking p105 only demonstrated elevated expression of

these members of the TNFSF. Nfkb1-/-

DCs had slightly higher (CD40, CD70) or equivalent

(OX40L) expression of these molecules without stimulation. Following stimulation with CpG,

Nfkb1-/-

DCs did not upregulate members of the TNFSF leaving them with equivalent (CD40,

CD70) or reduced (OX40L) expression compared to wild type DCs. In general, the effects of p50

and p105 on the expression of TNFSF molecules were mild in comparison to their effects on

MHC and B7 family molecules.

To determine the roles of p50 and p105 in regulating cytokine production in DCs, we

assayed TNFα, IL-6, and IL-12p70 in the supernatants from BMDC cultures stimulated or not

with CpG (Figures IV-1E). Nfkb1-/-

DCs failed to upregulate IL-12p70 production following

stimulation with CpG. By contrast, p105-/-

DCs have an enhanced ability to produce IL-12p70 in

response to stimulation. Both knockout DCs produced similar levels of TNFα as compared to

wild type DCs after CpG stimulation. However, Nfkb1-/-

DCs, but not p105-/-

DCs, had increased

basal expression of TNFα.

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Loss of p50 and p105 in DCs alters CD8+ T cell activation in vitro

In order to understand the mechanism by which Nfkb1-/-

and p105-/-

DCs induced altered T cell

activation, we examined T cell responses in vitro by coculturing LCMV-gp peptide pulsed

BMDCs with P14 TCR transgenic CD8+ T cells, which recognize the LCMV-gp33-41 peptide

in the context of H-2Db

[314]. Following 3 days of culture, P14 T cells were harvested and

analyzed for proliferation. In comparison to unstimulated wild type DCs, both Nfkb1-/-

and

p105-/-

unstimulated DCs induced increased P14 T cell proliferation, with p105-/-

DCs

stimulating the highest amount (Figure IV-2A). CpG stimulation increased the ability of wild

type, but not knockout DCs to induce P14 T cell proliferation such that they matched Nfkb1-/-

DCs while p105-/-

DCs still stimulated the most proliferation.

To test if our different DC populations had differential effects on T cell function, we

measured expression of granzyme B as a surrogate marker of cytotoxicity. Both Nfkb1-/-

and

p105-/-

unstimulated DCs induced elevated expression of granzyme B in P14 T cells in

comparison to wild type DC-stimulated P14s (Figure IV-2B, -2C). Wild type DCs matured with

CpG had a greatly enhanced ability to induce granzyme B expression. CpG-stimulated knockout

DCs were unable to upregulate granzyme B levels to similar levels as matured wildtype DCs. In

summary, both Nfkb1-/-

and p105-/-

DCs induced enhanced P14 T cell proliferation and granzyme

B production.

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Figure IV-2. CD8+ T cell activation is altered by loss of NFκB1 proteins. A. Isolated P14

cells were labeled with Cell Proliferation Dye and cultured for 3 days with IL-2 alone or LCMV-

gp33 pulsed BMDCs that had been stimulated or not with CpG ODN. Flow cytometry plots were

gated on CD8+ cells. B. P14 cells were stimulated as in A, followed by permeabilization and

intracellular staining of granzyme B. C. Quantification of geometric mean fluorescence of

markers analyzed in B, n=3. Statistical analysis for C was performed by two-way ANOVA with

Tukey’s test. Differences within treatment group are indicated, * p<0.05, ** p<0.01. Data are

representative of at least 3 experiments.

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Nfkb1-/-

DCs drive CD8+ T cell activation through antigen- and TNFα-dependent mechanisms

We next sought to further dissect the mechanism(s) driving the enhanced proliferation and

granzyme B expression observed in CD8+ T cells stimulated with knockout DCs. To determine

if the hyperproliferation induced by unstimulated Nfkb1-/-

DCs was dependent on cell-cell

contact, we assayed P14 T cells and DCs in transwell co-cultures (Figure IV-3A). P14 T cells

were cultured with wild type DCs in addition to wild type or Nfkb1-/-

DCs in the adjoining

chamber. There was no difference in the extent of P14 T cell proliferation between cells adjoined

with wild type or Nfkb1-/-

DCs, suggesting that the mechanism by which unstimulated Nfkb1-/-

DCs induce P14 T cell proliferation requires cell-cell contact.

Next, we asked if the ability of Nfkb1-/-

DCs to induce hyperproliferation of P14 T cells

requires cognate antigen presentation; that is, can Nfkb1-deficient DCs induce proliferation in T

cells receiving its TCR stimulation from another DC? To address this issue we co-cultured P14 T

cells with wild type DCs pulsed with LCMV-gp33, the cognate antigen for P14 T cells. To these

co-cultures we added Nfkb1-/-

DCs pulsed with adenovirus peptide (AV). DCs pulsed with AV

alone are unable to induce P14 T cell proliferation (Figure IV-3B). Furthermore, when added to

co-cultures of P14 T cells with wild type gp33-pulsed DCs, AV-presenting Nfkb1-/-

DCs were

only able to minimally increase P14 T cell proliferation, remaining significantly behind Nfkb1-/-

DCs pulsed with gp33. However, we observed a significant increase in the expression of

granzyme B when AV-presenting Nfkb1-/-

DCs were cultured alongside P14 T cells and gp33-

presenting wild type DCs (Figures IV-3C, -3D). Together these data suggests that distinct

mechanisms are driving P14 T cell hyperproliferation and granzyme B upregulation in response

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to Nfkb1-/-

DCs. While proliferation requires cognate antigen presentation, Nfkb1-/-

DCs appear to

be able to upregulate granzyme B in trans.

We previously demonstrated that both the upregulation of granzyme B in CD8+ T cells

and the induction of diabetes in RIP-gp mice in response to Nfkb1-/-

DCs is TNFα-dependent

[374]. We sought to determine what role TNFα played in our co-culture assay system. The

addition of a neutralizing TNFα antibody to DC:T cell co-cultures had no impact on the

proliferation of P14 cells (Figure IV-3E). However, neutralization of TNFα hindered the ability

of unstimulated Nfkb1-/-

DCs to induce granzyme B expression in P14 T cells (Figures IV-3F,

3G). However, TNFα neutralization had no impact on the expression of granzyme B in P14 T

cells cultured with CpG stimulated DCs, suggesting that another mechanism is driving granzyme

B expression following stimulation with CpG. In sum, both antigen- and TNFα-dependent

mechanisms are driving the enhanced activation of P14 T cells in response to Nfkb1-/-

DCs.

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Figure IV-3. Antigen- and TNFα-dependent mechanisms drive T cell activation by

NFκB1-deficient DCs. A. Isolated P14 T cells were fluorescently labeled with Cell Proliferation

Dye and cultured with LCMV-gp33 pulsed DCs in transwell culture plates in the indicated

configurations. The dotted line indicates the partition between the two chambers of the transwell.

Identity of “? DC” is indicated by the figure legend, either C57BL/6 (black line) or Nfkb1-/-

(orange line). Proliferation was measured after two days of culture by flow cytometry. B.

Fluorescently labeled P14 T cells were cultured with DCs for 3 days. DCs were pulsed with

LCMV-gp33 or AV peptide, and cultured in the indicated combinations. C. Cells were cultured

as in B, followed by permeabilization and intracellular staining for granzyme B. D. LCMV-gp33

pulsed DCs and fluorescently labeled P14 T cells were cultured for 3 days with or without the

addition of a neutralizing TNFα antibody. E. Co-cultures were setup as in D. On day 3, cells

were permeabilized and stained for intracellular granzyme B. All flow cytometry plots are gated

on CD8+ cells.

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Nfkb1-/-

DCs induce limited CD4+ T cell activation

As our analysis had thus far focused on CD8+ T cells, we next investigated the role of DC Nfkb1

in the activation of CD4+ T cells. In order to examine this issue, we utilized a co-culture assay

with Smarta TCR transgenic CD4+ T cells which recognize LCMV-gp61-81 in the context of I-

Ab [378]. Wild type and p105

-/- DCs induced similar levels of Smarta T cell proliferation (Figure

IV-4A). Nfkb1deficient DCs, however, were severely impaired in their ability to induce

proliferation of Smarta T cells. Additionally, we found that Smarta T cells stimulated with

Nfkb1-/-

DCs had a greatly reduced ability to produce the cytokines IL-2, TNF, and IFNγ in

comparison to both wild type and p105-/-

DCs (Figures IV-4B).

We had previously observed that Nfkb1-/-

DCs have greatly reduced expression of

MHCII. As such, we hypothesized that impaired expression of MHCII may be responsible for

the inability of Nfkb1-/-

DCs to induce normal CD4+ T cell activation. To test this hypothesis, we

performed co-culture experiments with plates coated with either αCD3 to compensate for the

lack of MHCII. The addition of αCD3 to the cultures restored proliferation in Nfkb1-/-

DC

stimulated Smarta T cells (Figure IV-4C). Furthermore, αCD3 was also able to restore

production of IL-2, TNF, and IFNγ in Smarta T cells (Figure IV-4D). These data suggest that

impaired MHCII expression in Nfkb1-/-

DCs is likely compromising their ability to induce

activation of CD4+ Smarta T cells.

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Figure IV-4. Nfkb1-deficient DCs fail to induce Smarta T cell activation. A. Smarta T cells

were fluorescently labeled and cultured with IL-2 or with LCMV-gp61 pulsed DCs for 3 days.

Proliferation was then measured by flow cytometry. B. Smarta T cells were cultured for 3 days

as in A. Cells were then restimulated with PMA/Ionomycin for 6 hours and treated with brefeldin

A for the final 4 hours. Intracellular staining for the indicated cytokines was then performed. C.

Tissue culture plates were coated with PBS or αCD3 cross-linked with an α-Hamster IgG. Co-

cultures were then carried out as in A.D. Smarta T cells were cultured as in C and restimulated

and stained as in B. All flow cytometry plots are gated on CD4+ cells.

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Loss of p105 in unstimulated DCs does not impart the ability to induce diabetes

To determine the in vivo immunogenicity of DCs lacking p50 and p105, we assayed them using

the RIP-gp model. Gp-peptide pulsed BMDCs were infused into RIP-gp mice and blood glucose

was monitored to detect the induction of diabetes (Figures IV-5A,-5B). Wild type DCs required

CpG stimulation in order to cause diabetes in RIP-gp mice. As previously reported [374], we

found that Nfkb1-/-

DCs were capable of inducing diabetes without CpG stimulation. CpG-

stimulated Nfkb1-/-

DCs also caused diabetes in RIP-gp mice (data not shown). By contrast,

unstimulated p105-/-

DCs were incapable of inducing diabetes. However, p105-/-

DCs stimulated

with CpG were able to induce diabetes, albeit with a slightly lower frequency than wild type DC.

These data suggest that the loss of p50 is a requisite event in allowing Nfkb1-/-

DCs to induce

diabetes in RIP-gp mice without CpG stimulation.

In order to better understand the immunogenicity of the DCs using this model, we

measured LCMV gp34-specific CD8+ T cells in the blood of RIP-gp mice, 6 days post

vaccination by tetramer staining (Figure IV-5C). In accordance with their ability to induce

diabetes, CpG-matured wild type DCs induced gp34 reactive T cells. By comparison, Nfkb1-/-

DCs also induced a similar proportion of gp34-reactive T cells in the absence of

CpG-stimulation. Despite their inability to induce diabetes, vaccination with unstimulated p105-/-

DCs resulted in a significant population of gp-34 reactive CD8+ T cells. This finding suggests

that p105-/-

DCs may, like Nfkb1-/-

DCs, are capable of inducing T cell expansion in vivo in the

absence of TLR maturation signals, but some other mechanism(s) are preventing the induction of

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CD8 mediated pathology. Together these data demonstrate that NFκB p50 is essential for

maintaining DCs in a non-immunogenic state.

Figure IV-5. Loss of p50 expression is required for unstimulated DCs to induce diabetes in

RIP-gp mice. A. BMDCs were stimulated overnight, pulsed for 3 hours with LCMV-gp

peptides, and infused into RIP-gp mice. Blood glucose was monitored over 2 weeks. Each line

represents one mouse. B. Mice with a blood glucose concentration of greater than 14mM were

scored as diabetic. Cumulative onset of diabetes in RIP-gp mice vaccinated with the indicated

DCs over multiple experiments, n=9-10. C. 6 days post vaccination, mice were bled and the

proportion of CD8+ T cells recognizing LCMV-gp34 was assayed by tetramer staining and flow

cytometry, n=3. Statistical analysis for C was performed by two-way ANOVA with Tukey’s test.

Differences within treatment group are indicated, * p<0.05. Data are representative of 2 (C) or 3

(A) independent experiments.

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Nfkb1 mixed bone marrow chimeras develop autoimmunity

In the RIP-gp model we observed that Nfkb1-/-

DCs, but not p105-/-

DCs, were able to activate

CD8+ T cells and trigger autoimmune responses without the requirement for TL-induced

maturation. This finding is sharply contrasted by the phenotype of Nfkb1-/-

and p105-/-

mice.

Nfkb1-/-

mice are immunocompromised and do not suffer from spontaneous inflammatory

pathologies [379]. By contrast, p105-/-

mice develop lymphoid hyperplasia and inflammatory

infiltrates in the lung and liver [313]. Given the spontaneous immunogenicity of Nfkb1-deficient

DCs, we questioned why Nfkb1-/-

mice do not develop autoimmune disease. As T cell responses

in Nfkb1-/-

mice are severely compromised [368] [369], we hypothesized that autoimmunity is

not observed in these mice despite the immunogenicity of Nfkb1-deficient DCs due to these

functional T cells defects.

To test this idea, we generated mixed bone marrow chimeras wherein irradiated wild type

hosts (Thy1.2+ CD45.1+) were reconstituted with either wild type (Thy1.1+ CD45.2+) or

Nfkb1-/-

(Thy1.2+ CD45.2+) bone marrow cells, or an equal mixture of the two (“mix”) (Figure

IV-6A). Mice that received Nfkb1-/-

bone marrow or a mixture of wild type and knockout bone

marrow developed splenomegaly (Figure IV-6B). This finding could largely be attributed to an

expansion of CD4+ T cells, while CD8+ T cells numbers were similar between groups. Using the

gating strategy indicated (Figure IV-6C), endogenous, wildtype donor, and knockout donor cells

could be differentiated. This approach revealed that CD4+ and CD8+ T cells in wild type and

mix recipients were largely donor derived (Figure IV-6D). By contrast, the CD4+ T cells in

Nfkb1-/-

recipients were approximately 50% host derived. This finding suggests that the CD4+ T

cell expansion found in knockout recipients is largely due to an expansion of host T cells.

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Figure IV-6. Nfkb1 bone marrow chimeras have altered T cell homeostasis. A. C57BL/6

mice were irradiated with 950 rad and then reconstituted with Thy1.1 bone marrow cells, Nfkb1-/-

bone marrow cells, or a 1:1 mixture of the two. Mice were then aged for 16 weeks before

analysis. B. Total number of splenocytes, splenic CD4+ and splenic CD8+ cells in bone marrow

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chimeras 16 weeks after reconstitution. CD4+ and CD8+ numbers are based on CD4/CD8 flow

cytometric profiling of splenocytes, n=4-5. C. Gating strategy for identification of cell origin.

Wild type donor cells were additionally gated for positive expression of Thy1.1. D. Proportion of

CD4+ and CD8+ splenocytes derived from the host (CD45.1+) in the indicated chimeric mice,

n=4-5. E. Expression of CD69 and CD44 on CD4+ splenocytes in bone marrow chimeras, as

determined by flow cytometry. F, G. CD4+ splenocytes, gated based on congenic markers as in

C, were analyzed for expression of CD69 and CD44 by flow cytometry. H,I. Quantification of

CD69 and CD44 expression from F, G, n=4-5. Statistical analysis was performed by one-way

ANOVA with Bonferroni’s post-test (B,D) or two-way ANOVA with Tukey’s test (H,I).

Statistical significance is indicated, * p<0.05, ** p<0.01. Data are representative of 2

independent experiments.

We then analyzed CD4+ and CD8+ T cells for expression of CD69, a marker of T cell

activation, as well as CD44, a marker of activated or memory-phenotype T cells. Both knockout

and mix recipients had an increase in the proportion of CD69+ and CD44

hi CD4+ T cells (Figure

IV-6E). By contrast, there were no significant differences in the number of CD8+ T cells with

elevated CD44 or CD69 expression between groups (data not shown). Elevated CD44 and CD69

expression on CD4+ T cells was uniquely found on wild type T cells, regardless of their origin

(Figures IV-6F, -6G, -6H, -6I). Although expression of CD44 and CD69 was not different on

total CD8+ T cells, we observed that expression of these molecules was higher on wildtype

T cells than on knockout T cells in “mix” and knockout chimeras. (Figure IV-6H, -6I).

In order to check for signs of autoimmune disease, we performed immunohistochemistry

on multiple organs of the chimeras. The liver, pancreas, lungs, and thyroid gland of mix and

knockout chimeras contained immune infiltrates while recipients of wild type marrow did not

(Figure IV-7A, 7B). While infiltrates often contained CD8+ cells, the bulk of the infiltration was

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due to CD4+ lymphocytes. Finally, to check for signs of system inflammation, we measured the

level of TNFα in the sera of chimeric mice (Figure IV-6C). In comparison to recipients of

wildtype cells, chimeras receiving bone marrow from both mix and knockout donors often had

elevated serum TNF, although this only reached statistical significance for mixed chimeras.

Together this suggests that wild type T cells are driving inflammation in bone marrow

chimeras containing Nfkb1-/-

hematopoietic cells; in mixed chimeras they are predominantly of

donor origin while in Nfkb1-/-

recipients, they are endogenous wild type T cells. These data

support the notion that compromised T cells in Nfkb1-/-

mice prevent the development of

autoimmunity.

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Figure IV-7. Nfkb1 bone marrow chimeras develop inflammation.

A. Immunohistochemistry for CD4 and CD8 of liver and lung from bone marrow chimeras. Bar

= 250µm. F. Cumulative fraction of organs with CD4 and/or CD8 infiltration from indicated

groups, n=8-10. G. Sera was isolated from chimeras 16 week post reconstitution and analyzed

for expression of TNFα by bead array. Each point represents one mouse. Statistical analysis for

C was performed by one-way ANOVA with Bonferroni’s post-test (C) or two-way ANOVA

with Tukey’s test (F,G). Statistical significance is indicated, * p<0.05, ** p<0.01. Data are

representative of 2 independent experiments.

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Discussion

NFκB p50 and p105 play distinct roles in DC biology

As Nfkb1 encodes for two proteins, p105 and p50, we have compared BMDCs from p105-/-

and

Nfkb1-/-

mice in order to dissect out the unique functions of each protein in DC biology.

Surprisingly, DCs generated from Nfkb1-/-

and p105-/-

had very distinct phenotypes. The loss of

p105 alone resulted in DCs that had enhanced expression of MHCII and B7 family members

CD80 and CD86. NFκB transcription factors are believed to drive expression of CD80 and CD86

[380]. It was previously demonstrated that DC expression of CD80 and CD86 was impaired by

combined loss of cRel and p50, while loss of RelA did not have an impact [381]. Therefore, the

enhanced expression of CD80 and CD86 observed in p105-/-

DCs may be the result of increased

NFκB activity. Moreover, inhibition of NFκB in DCs through the use of transduced IκBα

expression results in reduced CD80, CD86, and MHCII expression [382]. In response to TLR

stimulation, p105-/-

DCs produced elevated levels of IL-12, a known target of NFκB transcription

factors [383-385]. These findings, therefore, are consistent with reports that p105 functions as

an IκB protein, restraining the activity of NFκB transcription factors [107, 108].

Reports have also linked p105 with ERK signaling in DCs via the kinase tumour

progression locus-2 (TPL-2) [386]. In macrophages, p105 cleavage is required for activation of

ERK signaling following LPS stimulation [387, 388]. Furthermore, ERK signaling in DCs has

been demonstrated to inhibit expression of the master MHCII regulator CIITA [389]. Therefore,

it may be through the regulation of MAPK signaling that p105 restrains the expression of

MHCII.

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The phenotype of Nfkb1-/-

DCs was quite distinct from p105-/-

DCs, confirming that p50

plays an essential role in DC biology. In direct contrast to p105-/-

DCs, Nfkb1-/-

DCs had reduced

expression of MHCII, CD80, and CD86. Furthermore, Nfkb1 deficiency completely abolished

the ability of DCs to upregulate these molecules following stimulation with CpG. This finding is

consistent with previous reports of p50 containing heterodimers activating pro-inflammatory

genes downstream of TLR signaling [381, 390]. Additionally, Nfkb1-deficient DCs were

impaired in their ability to produce IL-12p70 and IL-6 in response to CpG. These data reinforce

suggestions that p50:cRel and/or p50:RelA heterodimers promote transcription of IL-12 [384,

385, 391]. TNFα production following TLR signaling has also been suggested to involve p50-

containing heterodimers [392, 393], however, we did not detect impaired TNFα production

following CpG stimulation.

We also confirmed our earlier finding that the loss of Nfkb1 in DCs leads to increased

basal TNFα production [374]. This phenomenon was not observed in p105-/-

DCs, suggesting

that p50 is critical for preventing spontaneous TNFα release. NFκB p50 lacks a transactivation

domain [394]. Therefore, the p50 homodimers which can be found in various resting cell types

are believed to be transcriptionally repressive [394, 395]. Notably, suppression of TNFα

production has been previously suggested to be mediated by p50 homodimers, particularly in the

context of endotoxin resistance [370, 396]. Together this suggests that the lack of p50

homodimers in Nfkb1-deficient DCs may facilitate basal expression of TNF.

This study aimed to dissect out the distinct roles of p50 and p105 in DC biology. We

have clearly demonstrated p105-specific functions in restricting the expression of inflammatory

cytokines and co-stimulatory molecules, most noticeably following CpG stimulation. We

identified a dramatically different phenotype for Nfkb1-/-

DCs. Many of the effects observed in

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Nfkb1-/-

DCs are consistent with previous reports on the function of p50 homodimers as

transcriptional repressors. However, our study cannot distinguish between the effect of loss of

p50 and the effect of combined loss of p50 and p105 on DC biology. For example, it may be that

basal transcription of TNFα requires loss of both p50 and p105. One strategy which has been

used to further deduce the intrinsic functions of p50 and p105 is the use of Nfkb1 mutant mice.

Nfkb1-SSAA mice harbor serine to alanine mutations in p105 which prevents its IKK-mediated

phosphorylation and subsequent proteolytic cleavage into p50 [397]. These mice have been used

to demonstrate important roles for the cleavage of p105 into p50 during T cell [397] and B cell

[398] activation. The use of Nfkb1-SSAA mice may help to further differentiate the individual

roles of p50 and p105 in regulating DC biology.

DC expression of NFkB p50 is required to prevent CD8+ T cell-mediated pathology

Steady state DCs are tolerogenic and are believed to only transform into an immunogenic state

following their maturation. Mounting evidence indicates that this non-immunogenic state is

actively maintained by various intrinsic factors [363]. We previously demonstrated that Nfkb1 is

a regulatory factor that maintains DC quiescence, without which DCs spontaneously become

immunogenic [374]. We have now demonstrated that of the two Nfkb1 encoded proteins, p50 is

required to maintain DC homeostasis; loss of p105 alone is insufficient to confer to DCs the

ability to induce CD8+ T cell-mediated pathology in the RIP-gp model. This finding may be

surprising given the individual phenotypes of Nfkb1-/-

and p105-/-

DCs. The loss of p105 in

unstimulated DCs resulted in a DC phenotype that more closely resembled that of a classic TLR

stimulated DC, with increased expression of MHCII, CD80, CD86, and various TNFSFRs.

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However, only Nfkb1-/-

DCs, which have depressed expression of MHCII, CD80, and CD86,

were able to induce immune-mediated diabetes. This finding is contrasted by our in vitro

experiments which demonstrated that p105-/-

DCs induced more proliferation, more cytokine

production, and equivalent granzyme B expression in P14 cells as compared to Nfkb1-/-

DCs.

Furthermore, in agreement with our co-culture experiments, unstimulated p105-/-

DCs were able

to induce expansion of gp34-reactive T cells, and were unable to sufficiently induce an

inflammatory CD8+ T cell response that resulted in immune pathology.

One critical feature of Nfkb1-/-

DCs that p105-/-

DCs may be lacking is constitutive

secretion of TNF. We have demonstrated its clear role in regulating granzyme B expression in

CD8+ T cells during activation by unstimulated DCs. Despite this lack of TNFα secretion,

unstimulated p105-/-

DCs also induced elevated granzyme B expression in P14 cells. Although

not yet formally tested, this strongly suggests that p105-/-

DCs may be using an alternative

mechanism to induce granzyme B. TNFα can have co-stimulatory properties for CD8+ T cells,

particularly in the context of limited inflammatory signals such as in an anti-tumour setting [399-

401]. Although we have demonstrated that TNFα is playing a direct role in activating CD8+ T

cells, it may have additional functions in vivo which contribute to the immunogenicity of Nfkb1-/-

DCs. For example, TNFα is known to contribute to lymph node remodeling during inflammation

[402]. Regardless, we know that TNFα signals are essential for the induction of diabetes in RIP-

gp mice by Nfkb1-/-

DCs, as Nfkb1-/-

DCs were unable to induce diabetes in RIP-gp TNFR1-/- or

RIP-gp/ TNFR2-/- mice [374].

We have additionally determined that Nfkb1-/-

DCs use a TNF-independent mechanism to

accelerate CD8+ T cell proliferation. This mechanism was dependent on presentation of cognate

antigen. One possibility is that this is related to the elevated expression of MHCI. Alternatively,

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this phenomenon may depend on the tight and prolonged contacts that form during antigen

presentation [403]. If this is the case, given the low expression of co-stimulatory ligands on

Nfkb1-/-

DCs, the molecules evolved in this process remain elusive.

Nfkb1 chimerism disrupts T cell homeostasis and promotes inflammation

NFκB p50 homodimers have reported roles in restricting inflammation [367]. In spite of

this, Nfkb1-/-

mice do not develop overt autoimmune disease [379]. One explanation for this

finding is that Nfkb1 encodes for both pro- and anti-inflammatory functions. Loss of the anti-

inflammatory properties of p50 homodimers and IκB functions of p105 may be sufficiently

countered by loss of pro-inflammatory effects of p50-containing NFκB heterodimers. This could

occur in a cell-intrinsic manner. Alternatively, distinct pro- and anti-inflammatory functions of

Nfkb1 in different cell types may collude to maintain tolerance.

We have demonstrated that the mixed presence of Nfkb1sufficient and deficient

hematopoietic cells disrupts immune homeostasis, resulting in the induction of multi-organ

autoimmunity including CD4+ T cell dominant immune infiltration. Furthermore, we found that

only wild type T cells are able to be activated in this setting, confirming previous studies that

have demonstrated the compromised function of Nfkb1deficient T cells [368]. Although we have

demonstrated a clear role for DC p50 in limiting CD8+ T cell activation, Nfkb1deficient DCs are

impaired in their ability to directly active CD4+ T cells. Therefore, it seems unlikely that

Nfkb1deficient DCs are the sole cell population driving autoimmunity in these chimeras. TNFα is

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also known to be co-stimulatory for CD4+ T cells [404]. One possibility then is that constitutive

TNFα secretion in the context of normal antigen presentation, such as by wildtype APCs, may

lead to CD4+ T cell activation. DCs themselves also respond to TNFα through upregulation of

co-stimulatory molecules. It may be then that TNFα is driving the maturation of wildtype DCs,

promoting activation of CD4+ T cells. Remarkably, p105-/-

mice develop CD4+ immune

infiltrates primarily in the lung and liver [313], the two organs most severely affected in our bone

marrow chimeras. This finding, perhaps, points to a common mechanism driving autoimmunity

in Nfkb1 chimeras and p105-/-

mice.

Interestingly, deregulated Nfkb1 control of TNFα production may contribute to human

autoimmune disease. A polymorphism in the TNFα promoter (-863A) has been identified which

results in a reduced ability of p50 homodimers to bind and block TNFα transcription [405]. This

polymorphism has been found to be associated with various autoimmune diseases [406-408].

Altogether these findings suggest that Nfkb1 plays an important in preventing the development of

autoimmunity.

Concluding Remarks

The maintenance of steady state DCs is crucial for maintaining immune tolerance. Nfkb1 is an

essential component of the quiescent DC program. We have herein demonstrated that the loss of

p50 in Nfkb1-/-

DCs is an essential event that potentiates their ability to induce T cell pathology

in vivo. Without p50, DCs constitutively secrete TNFα and are capable of inducing pathogenic

CD8+ T cell responses. In contrast to its role in immature DCs, p50 appears to play an important

role in TLR-stimulated DC activation; DCs lacking p50 are severely impaired in their ability to

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express the cytokines and surface molecules traditionally associated with DC immunogenicity.

Future studies will be required to closely examine the molecular events in Nfkb1 DCs which are

facilitating their production of TNFα and activation of T cell proliferation. A more thorough

understanding of this dyregulated DC activation may provide insights into the development of

sterile immune responses which may lack strong PRR ligands to activate DCs, such as in

autoimmunity and cancer.

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Chapter V

A20 and the Molecular Regulation of NFκB

Dylan Johnson1,2

, Wenxin Chen2, Carlos Garcia-Batres2, Celine Robert-Tisso

2, Pamela

Ohashi1,2

1Department of Immunology, University of Toronto, Toronto, Canada

2Campbell Family Institute for Breast Cancer Research, Toronto, Canada

All authors contributed to the design of experiments.

DJ performed experiments with assistance from WC, CR, and CG.

CG performed RT-PCR analysis.

DJ and PO wrote the manuscript.

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Introduction

The NFκB transcription factors are ubiquitous regulators of immune homeostasis and activation.

NFκB signaling has well established roles in inflammation, promoting the differentiation and

activation and T cells, B cells, DCs, macrophage, amongst others [66]. Additionally, it is now

appreciated that NFκB plays important roles in maintaining tolerance. For example, NFκB, and

in particular cRel, have been demonstrated to be important for the development of Tregs [409-

412]. Furthermore, loss of NFκB family members Relb or Nfkb2 results in impaired development

of thymic medullary epithelial cells and consequently impaired negative selection and

autoimmunity [413, 414]. The NFκB subunit p50 is known to mediate endotoxin resistance [370,

396]. Moreover, we have suggested a role for p50 in preventing spontaneous DC activation

(Chapter IV, [374]). Proper molecular regulation of NFκB is therefore paramount to both the

maintenance of tolerance and production of immunity.

Several distinct mechanisms are known to regulate the activity of NFκB. By far the most

widely known is the sequestration of NFκB dimers by IκB proteins. In resting immune

cells, NFκB dimers are sequestered in the nucleus where they are bound to IκB by virtue of their

ankyrin-repeat domains [90]. Following cell activation, IκB proteins are phosphorylated and

subsequently degraded, resulting in nuclear translocation of NFκB proteins and activation of

transcription. IκB proteins are also an essential negative feedback mechanism that limits the

duration of NFκB signaling. For example, the transcription of IκBα is driven by NFκB and

therefore its expression is upregulated following NFκB activation. Disruption of this mechanism

through mutation of the κB sites in the IκBα promoter leads to disrupted T cell homeostasis and

the development of autoimmunity [415].

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It is also clear that ubiquitin-modifying enzymes play an important role in the regulation

of NFκB signaling [416]. E3 ligases such as TRAF2 and TRAF6 have well-known roles in

activating NFκB signaling following stimulation [417]. It has further become apparent that

deubiquitinases are also critical for regulating NFκB [418]. Of these, CYLD and A20 have been

the best studied. CYLD has been shown to remove K-63-linked ubiquitin chains from TRAF2,

TRAF6, and NEMO, thereby terminating NFκB signaling [151-153]. A20 is unique in that it

possess enzymatic domains to catalyze both the hydrolysis of K63-linked polyubiquitin chains

and the addition of K48-linked chains [419]. The NFκB signaling intermediates TRAF2, TRAF6,

and RIP1 have all been identified as targets of A20 [160, 167, 420]. The critical role of A20 in

limiting pro-inflammatory role is clearly demonstrated by the fatal inflammation suffered by

A20-knockout mice as well as GWAS studies which have linked A20 polymorphisms with

autoimmune diseases such as Crohn’s disease, rheumatoid arthritis, and systemic lupus

erythematous [421].

Recent work has identified a cell-intrinsic role for A20 in maintaining DC homeostasis

[187, 188]. Conditional deletion of A20 results in elevated DC expression of CD40, CD80,

CD86, MHC class II, and IL-6 as well as a compromised ability to maintain immune tolerance.

One study reported that conditional deletion of A20 from DCs resulted in systemic

autoimmunity, including the generation of anti-dsDNA autoantibodies [188]. By contrast,

another report found that A20 conditional knockout mice developed autoimmune disease that

was limited to colitis and ankylosing arthritis, and did not produce autoantibodies [187]. Given

the severe and complex phenotype of A20 conditional knockout mice, it remains unclear the

precise cellular mechanisms by which A20-deficient DCs are promoting the loss of tolerance.

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Furthermore, the consequence of A20 deficiency on the ability of DCs to specifically regulate

CD8+ T cell responses has not been established.

Our laboratory has previously developed a vaccination model to test the ability of DCs to

induce CD8+ T cell-mediated tissue destruction [374]. We therefore seek to determine what role

DC expression of A20 has in controlling CD8+ T cell immunity using this model. Additionally,

we have previously identified Nfkb1 as another important cell-intrinsic factor restricting

functional DC maturation (Chapter IV, [374]). Through comparison of Nfkb1 and A20-

deficiency, we aim to delineate a molecular pathway that regulates steady state DCs.

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Results

Generation of A20 deficient dendritic cells

To determine the role of A20 in regulating DC biology, we generated mice homozygous for a

floxed allele of TNFaip3 (A20fl/fl

) that also expressed Cre recombinase under the control of the

Itgax promoter (CD11c-cre). We generated BMDCs from A20fl/fl

(wild type) and A20fl/fl

CD11c-

cre (conditional knockout) mice by culturing bone marrow cells in the presence of GM-CSF for

8-10 days. Bone marrow from both wildtype and conditional knockout mice generated a majority

of CD11c CD11b cells, demonstrate that A20 is not required for in vitro differentiation of DCs

(Figure V-1A).

A20 is required for conventional dendritic cell maturation

We next sought to determine what effect A20 deficiency had on the regulation of DC cell-

surface molecules. DCs were cultured overnight with or without CpG and then analyzed by flow

cytometry (Figures V-1B, -1C). Compared to wildtype cells, A20 knockout DCs had slightly

elevated expression of MHCI. By contrast, conditional knockout cells had marginally lower

MHCII and impaired upregulation of MHCII in response to CpG. A similar trend was found in

the expression of B7 family molecules. Both CD80 and CD86 had reduced expression on A20-

deficient DCs. Furthermore, the ability to upregulate CD80 and CD86 in response to CpG was

completely abolished by A20 deficiency. We additionally examined members of the TNFSF,

CD40, CD70, and OX40L. We did not observe any notable differences in the expression of these

molecules between wildtype and conditional knockout DCs.

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We also examined the role of A20 in regulating cytokine production in DCs.

Supernatants from overnight DC cultures were assayed for IL-12p70, IL-6, TNFα, MCP-1, and

IL-10 by bead array (Figure V-1D). Loss of A20 expression resulted in elevated secretion of

IL-12p70, IL-6, MCP-1, and IL-10 in response to CpG stimulation. A20-deficient DCs

constitutively expressed TNFα at low levels. However, following CpG stimulation, loss of A20

resulted in impaired TNFα production. In summary, A20-deficiency results in impaired

expression of many DC maturation markers (MHCII, CD80, CD86) and enhanced expression of

many inflammatory cytokines (IL-12p70, IL-6, MCP-1) in response to CpG while also limiting

basal TNFα expression.

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Figure V-1. Generation of A20-deficient DCs. A. Bone marrow cells from A20fl/fl

, and A20fl/fl

CD11c-cre mice were cultured for 8-10 days in GM-CSF to generate BMDCs. Expression of

CD11b and CD11c was measured by flow cytometry. B BMDCs, with or without overnight

stimulation with CpG, were stained with monoclonal antibodies against the indicated markers

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and analyzed by flow cytometry. Plots are gated on CD11chi

Cd11bhi

events as in A. C.

Quantification of geometric mean fluorescence of markers analyzed in B, n=3. D. Following

overnight stimulation, supernatants from BMDC cultures were collected and analyzed by bead

array for the presence of the indicated cytokines, n=3. Statistical analyses for C and D were

performed by two-way ANOVA with Sidak’s multiple comparisons test. Differences within

treatment groups are indicated, * p<0.05, ** p<0.01. Data are representative of at least 3

independent experiments.

Loss of A20 in DCs has minor impact on in vitro T cell activation

To elucidate any functional differences between wild type and A20 knockout DCs, we examined

early events in T cell activation in vitro (as described in Chapter IV). Briefly, LCMV-gp33

specific P14 T cells were cultured for 3 days with gp33-pulsed DCs. Unstimulated A20 knockout

DCs induced a minor increase in P14 T cell proliferation in comparison to unstimulated wild

type DCs (Figure V-2A). CpG-stimulation of wildtype DCs increased P14 T cell proliferation.

However, P14 cells cultured with CpG-stimulated wild type and knockout DCs proliferated to

the same extent.

To test the consequence of A20 loss on the ability of DCs to induce T cell cytotoxicity,

we measured granzyme B expression in P14 T cells following coculture with wild type or A20

knockout DCs (Figure V-2B). In comparison to an IL-2 alone control, Granzyme B expression

was upregulated by unstimulated DCs. Granzyme B expression was further enhanced by CpG-

stimulation of DCs. We detected no differences in the ability of wild type and A20-deficient

DCs, with or without stimulation, to induce granzyme B expression in P14 T cells.

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Next, we determined whether role DC A20 has a role in regulating cytokine production in

T cells. After 3 days in culture with DCs, we restimulated P14 T cells with PMA/ionomycin and

measured cytokine production by intracellular staining. We found no differences in the abilities

of wild type and A20 knockout DCs to induce production of IL-2, TNFα, or IFNγ in T cells

(Figures V-2C, -2D). Together, these data suggest that loss of A20 has only a minor impact on

the ability of DCs to induce CD8+ T cell activation in vitro.

Figure V-2. Dendritic cell A20 has minimal impact on in vitro P14 activation. A. P14 T cells

were fluorescently labeled with Cell Proliferation Dye and then cultured for 3 days at a 10:1 ratio

with BMDCs pulsed with LCMV-gp33. Proliferation was analyzed by fluorescence dilutions

using flow cytometry. B. P14 T cells were cultured as in A followed by intracellular staining for

granzyme B. C. P14 T cells were cultured as in A. Cells were then stimulated with

PMA/Ionomycin for 6 hours and treated with brefeldin A for the final 4 hours. Expression of the

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indicated cytokines was then determined by intracellular staining and flow cytometry. D.

Quantification of intracellular cytokine staining as performed in C, n=3. E. Supernatants from

P14:BMDCs co-cultures were analyzed for the presence of the indicated cytokines by bead array,

n=3. All flow cytometry plots are gated on CD8+ events. Statistical analysis for D and E was

performed by two-way ANOVA and Sidak’s multiple comparisons test. Differences within

treatment group are indicated, * p<0.05, ** p<0.01. Data are representative of at least 3

experiments.

A20 maintains DC quiescence

To determine the impact of loss of A20 on the ability of DCs to induce CD8 effector T cell

responses in vivo, we assayed them with the RIP-gp model as previously described (Chapter IV).

Briefly, RIP-gp mice were vaccinated with BMDCs pulsed with LCMV-gp peptides. The

induction of a potent CD8+ T cell response against gp in RIP-gp results in pancreatic β-islet

destruction and consequently, diabetes. Unstimulated wild type DCs were unable to induce

diabetes in RIP-gp mice (Figure V-3A, 3B). As previously shown, CpG matured DCs were able

to induce a functional CD8+ T cell response leading to diabetes [366]. By contrast, vaccination

of RIP-gp mice with unstimulated or CpG-stimulated A20-deficient DCs resulted in the

induction of diabetes. This demonstrates that A20 is required to maintain steady state DCs.

To further examine whether A20 knockout DCs were able to induce CD8 T cell

responses in vivo, we measured the expansion of gp34-reactive T cells using tetramer staining 8

days post infusion. DC vaccination resulted in an expansion of CD8+ gp34-reactive T cells in

both the spleen and pancreatic-draining lymph node (pdLN) (Figure V-3C). CpG stimulation of

wild type DCs resulted in a greater expansion of gp34 reactive CD8+ T cells in both the spleen

and pdLN. However, there were no significant differences in ability of wild type or A20

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knockout DCs to promote T cell expansion. Therefore, the ability of A20-deficient DCs to

induce diabetes cannot be explained by differential induction of T cell proliferation.

We next measured the cytotoxic T cell response induced by vaccination with wild type

and A20-deficient DCs using an in vivo cytotoxicity assay. C57BL/6 mice were vaccinated with

LCMV-gp peptide pulsed DCs. 6 days post vaccination, the mice were infused with a 1:1

mixture of fluorescently-labeled splenocytes that had been pulsed with either LCMV-gp33 or

with the control adenovirus peptide (AV). Recipient spleens were harvested 5 hours later and the

specific loss of g33 pulsed splenocytes is reported as cytotoxicity. Unstimulated wild type DCs

induced very little cytotoxicity (Figure V-3D). Wild type DCs stimulated with CpG had an

increased ability to induce cytotoxicity. Unstimulated A20 knockout DCs induced an enhanced

cytotoxic response that was even greater than that induced by CpG-stimulated wild type DCs.

Additionally, CpG-stimulated A20 knockout DCs induced very high levels of cytotoxicity.

Together, these findings demonstrate that A20 is critical for maintaining steady state DCs. The

absence of A20 results in a DC that is able to induce a superior cytotoxic response in vivo, even

in the absence of TLR-induced maturation.

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Figure V-3. Unstimulated A20-deficient DCs induce diabetes in RIP-gp mice. A. BMDCs

were stimulated overnight, pulsed for 3 hours with LCMV-gp peptides, and infused into RIP-gp

mice. Blood glucose was measured to monitor for the onset of diabetes. Each line represents a

single mouse. B. Mice with a blood glucose concentration of greater than 14mM were scored as

diabetic. Cumulative onset of diabetes in RIP-gp mice vaccinated with the indicated DCs over

multiple experiments, n=9. C. 6 days after DC infusion, animals were sacrificed and spleen and

pancreatic-draining lymph node (pdLN) cells were harvested and stained. Reported is the

proportion of CD8+ T cells with positive staining for an LCMV-gp34 tetramer, n=3. D.C57BL/6

mice were vaccinated with BMDCs, prepared as in A. 6 days post vaccination, mice were

infused with a 1:1 mixture of fluorescently labeled splenocytes pulsed with LCMV-gp peptides

or with adenovirus peptide (AV). Bars indicate the proportional loss of LCMV-gp-pulsed

splenocytes over AV-pulsed splenocytes, as compared to an unvaccinated control mouse, n=3.

Statistical analysis for B was performed by log-rank test. Statistical analysis for C and D was

performed by two-way ANOVA and Sidak’s multiple comparisons test. Differences within

treatment group are indicated, * p<0.05, ** p<0.01. Data are representative of at least 3

experiments.

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The expression of NFκB proteins is ablated in Nfkb1- and A20-deficient DCs

A20 is a known negative regulator of NFκB signaling and NFκB activation mediates DC

maturation [160]. We therefore hypothesized that increased NFκB activity may account for the

immunogenicity of A20-deficient DCs. We examined the expression of NFκB proteins in wild

type and A20-deficient DCs by Western blot (Figure V-4A). Surprisingly, the expression of

NFκB transcription factors (p50, p65, cRel, relB), inhibitors of NFκB (IκBα, IκBβ), and the

NFκB p50 precursor protein p105 was dramatically reduced in A20-deficient DCs.

We have previously demonstrated that Nfkb1, like A20, is required to maintain DCs in a

quiescent state, as demonstrated using the RIP-gp model (Chapter IV, [374]). In addition, both

Nfkb1 proteins p50 and p105 are thought to play regulatory roles in NFκB signaling. We

therefore examined Nfkb1-/-

DCs for the expression of NFκB to see if they also displayed this

unexpected molecular phenotype. Nfkb1-/-

DCs had severely reduced expression of p65, cRel,

RelB, IκBα, and IκBβ (Figure V-4B). To confirm the loss of the NFκB signaling axis in Nfkb1-/-

DCs, we performed an NFκB electromobility shift assay (EMSA) on cytoplasmic and nuclear

extracts (Figure V-4C). Nuclear wild type extracts had the ability to bind and shift κB consensus

DNA which was enhanced through stimulation with CpG. By contrast, nuclear extracts from

unstimulated and CpG-stimulated Nfkb1-/-

DCs were unable to induce a shift, confirming the loss

of NFκB activity. However, we noted that overnight culture of Nfkb1-/-

DCs with CpG resulted in

a partial restoration of some NFκB proteins, namely p65, cRel, and IκBβ (Figure V-4B).

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Figure V-4. A20 and Nfkb1-deficient DCs have reduced expression of NFκB proteins. A.

BMDCs derived from A20fl/fl

and A20fl/fl

CD11c-cre mice were lysed and probed for the

expression of the indicated NFκB proteins by Western blot. B. After overnight culture with or

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without CpG, BMDCs were lysed and analyzed for the expression of the indicated NFκB

proteins by Western blot. C. Wild type or Nfkb1-/-

BMDCs were stimulated or not with CpG for

1 hour. Cytoplasmic and nuclear fractions were then extracted from the BMDCs. Extracts were

analyzed by EMSA using consensus κB DNA. D. Following overnight culture with or without

CpG, RNA was isolated from wild type or Nfkb1-/-

BMDCs and submitted to qPCR analysis of

the indicated genes. E. A20fl/fl

and A20fl/fl

CD11c-cre BMDCs were analyzed as in D. Error bars

indicate the standard deviation of the ΔΔCT. F. Splenic DCs were isolated from the indicated

mice and immediately lysed and analyzed by Western blot for the indicated proteins.

To test if the loss of NFκB expression in A20- and Nfkb1-deficient DCs was the result of

transcriptional regulation, we examined the expression of NFκB mRNA by qPCR. Unstimulated

Nfkb1-/-

DCs had reduced levels of Nfkb1 (p105, p50), Nfkb2 (p100, p52), Rela (p65), Relb

(RelB), and Rel (cRel) mRNA (Figure V-4D), suggesting altered transcriptional regulation of

these genes. The detection of Nfkb1 mRNA in Nfkb1-/-

DCs reflects the fact that the deletion

cassette is inserted into exon 6 of Nfkb1 while our qPCR primers detect exon 1. This finding then

suggests that the transcriptional program of Nfkb1-deficient DCs is primed for reduced Nfkb1

transcription as is the case for the other NFκB genes.

Despite the ability of CpG-stimulation to restore NFκB protein expression in Nfkb1-

deficient DCs, stimulation did not significantly alter mRNA expression. A20-/-

DCs displayed an

analogous phenotype; expression of Nfkb1, Nfkb2, Rela, Relb), and Rel was significantly reduced

compared to wildtype DCs and was not significantly altered by CpG stimulation (Figure V-4D).

Although NFκB mRNA levels are consistently reduced in A20 and Nfkb1-deficient DCs, their

expression is often approximately half of wild type levels. This suggests that transcriptional

regulation alone cannot account for the complete loss of NFκB expression observed in knockout

DCs.

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In contrast the depressed expression of NFκB genes, we found that expression of Nfkbia

was elevated in A20-/-

DCs (Figures V-4D, -4E). As IκBα protein levels were severely reduced in

A20-/-

DCs, this finding strongly suggests that post-transcriptional mechanisms are responsible

controlling the levels of IκBα.

We had thus far explored this unexpected molecular phenotype with in vitro generated

BMDCs. To verify that this phenotype was also true of endogenous DCs, we examined the

expression of NFκB proteins in splenic DCs (Figure V-5B). Nfkb1-/-

DCs isolated from the

spleen expressed p65, suggesting that endogenous DCs in Nfkb1-/-

mice do not display this

molecular phenotype. By contrast, splenic A20-/-

DCs had sharply reduced expression of both

p65 and p50 demonstrating that the loss of NFκB proteins can also occur in DCs in vivo.

A20- and Nfkb1-deficient DCs both constitutively express low levels of TNFα

(Figures IV-I, V-I,[374]). Expression of TNFα can be driven by NFκB transcription factors

[393]. Our observations, however, strongly suggest that NFκB is not responsible for driving

TNFα production in these DCs. We therefore sought to examine the status of other transcription

factors which may be driving the expression of TNFα. AP-1 can drive TNFα expression and is

known to regulate DC maturation [393, 422]. We therefore examined AP-1 activity in Nfkb1-/-

DCs by EMSA. Nuclear extracts from unstimulated Nfkb1-/-

DCs demonstrated less AP-1

consensus DNA binding than did wild type extracts. This finding suggests that AP-1 activation is

not responsible for basal TNFα production in Nfkb1-/-

DCs. We went on to examine the

expression of other transcription factors which are known to drive expression of TNFα:

interferon response factors (Irf-1, -3, and -8)[423-425] and erythroblast-transformation specific-1

(Ets1)[426]. We found equivalent or slightly reduced expression of all of these transcription

factors in unstimulated Nfkb1-/-

DCs (Figure V-5B). Likewise, there was no increased expression

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of Ets1 in A20-deficient DCs (Figure V-5C). Together these data do not suggest roles for AP-1,

Ets1, or Irf proteins in transcription of TNFα in knockout DCs. Future biochemical studies of the

TNFα promoter in A20 and Nfkb1 knockout DCs will be required to characterize the

transcription factor(s) driving expression of TNFα.

Figure V-5. Nfkb1-deficient DCs do not have elevated expression of TNFα transcription

factors. A. Wild type or Nfkb1-/-

BMDCs were cultured with or without CpG for 1 hour.

Cytoplasmic and nuclear fractions were then extracted from the BMDCs. Extracts were analyzed

by EMSA using consensus AP-1 DNA. B. Following overnight culture with or without CpG,

RNA was isolated from wild type or Nfkb1-/-

BMDCs and analyzed by qPCR for the indicated

genes. C. A20fl/fl

and A20fl/fl

CD11c-cre BMDCs were analyzed as in B. Error bars indicate the

standard deviation of the ΔΔCT

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Inhibition of the proteasome partially restores NFκB protein expression in Nfkb1-deficient DCs

The loss of NFκB proteins in A20- and Nfkb1-deficient DCs did not appear to be solely a

consequence of altered transcription. Experiments were performed to evaluate whether post-

translational regulation was contributing to this phenotype. To test if protein degradation was

responsible for the loss of NFκB we treated our DCs with two protease inhibitors, lactacystin and

MG-132. Treatment with lactacystin resulted in a slight increase in p65 expression in Nfkb1

knockout DCs (Figure V-6A). However, neither cRel nor IκBα expression was altered by

lactacystin. When NFκB1-deficient DCs were treated with MG-132, we observed increased

expression of relB, p65, cRel, and IκBα (Figure V-6B). This increase was present within 1 hour

of treatment with MG132 and the effect also appeared to be maximal at this time point.

Protein degradation is often directed by the activities of E3 ligases which add K48-linked

polyubiquitin chains to proteins, targeting them to the proteasome. Several components of E3

ligases complexes, including PDZ and LIM domain-2 Mystique (PDLIM2), suppressor of

cytokine signaling-1 (Socs1), and copper metabolism domain-containing-1 (COMMD1), have

been previously suggested to target NFκB proteins [136, 137, 427]. To determine if these

proteins may play a role in regulating NFκB proteins in A20- and Nfkb1-deficient DCs, we

examined their expression by qPCR (Figures V-6C, D). Both A20- and Nfkb1-deficient DCs

expressed much more Socs1 mRNA in comparison to wild type DCs. The expression of Commd1

and Pdlim2 was equivalent in Nfkb1 knockout DCs and slightly reduced in A20 knockout DCs.

This data suggests that upregulation of SOCS1 could contribute to the molecular phenotype

observed in knockout DCs.

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In summary, we have demonstrated that reduction of NFκB proteins is observed in steady

state DCs that do not express A20 or Nfkb1. This phenotype results from both transcriptional

regulation and active protein degradation.

Figure V-6. Proteasome inhibition results in partial restoration of NFκB proteins in

Nfkb1-deficient DCs. A. Wild type or Nfkb1-/-

BMDCs treated for the indicated number of hours

with lactacystin. BMDCs were then lysed an analyzed by Western blot for the expression of the

indicated proteins. B. As in A but with MG-132 treatment. C. Following overnight culture with

or without CpG, RNA was isolated from wild type or Nfkb1-/-

BMDCs and submitted to qPCR

analysis of the indicated genes. D. A20fl/fl

and A20fl/fl

CD11c-cre BMDCs were analyzed as in C.

Error bars indicate the standard deviation of the ΔΔCT

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Discussion

A20 is required to preserve DC quiescence

Previous reports have identified A20 as a critical cell-intrinsic regulator of DC homeostasis [187,

188]. The loss of A20 specifically in DCs results in the splenomegaly and lymphadenopathy,

driven by the expansion of T cells, B cells, macrophage, and DCs [187]. Furthermore, T cells in

these mice display an activated/effector phenotype, demonstrating the crucial role that

appropriate regulation of DC maturation plays in preserving T cell homeostasis. Furthermore, the

conditional loss of A20 in DCs leads to the development of autoimmune disease.

While both reports on the function of A20 in DCs report the development of

autoimmunity, each group found distinct autoimmune manifestations. Hammer et. al. report the

development of T cell-driven colitis and spondyloarthritis [187]. By contrast, Kool et. al.

observed a Lupus-like disease including the production of anti-dsDNA antibodies and nephritis

[188]. Both groups performed experiments with C57BL/6 background mice and used the same

CD11c-cre to drive deletion [428]. One key difference between the experiments of Kool and

Hammer is the exact nature of the knockout used. The floxed Tnfaip3 allele used by Kool et. al.

led to the excision of exons 4 and 5. Hammer et. al. used a Tnfaip3 allele that excises exon 2

from A20. The OTU domain of A20, which catalyzes deubiquitination of target proteins, is

contained within exon 2 [164]. It is therefore possible that residual A20 deubiquitinases function

in the mice of Kool et. al. may have contributed to the differences observed between the two

studies. Alternatively, differences between the microbiota of the animals may have also played a

role. The microbiota has been demonstrated to influence the development of autoimmune disease

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[429]. Furthermore, Hammer et. al. demonstrated that the development of lymphadenopathy was

dependent on MyD88, further suggesting a role for the microbiota in the development of

autoimmune disease in conditional A20 knockout mice.

We have herein confirmed a cell-intrinsic role for A20 in regulating DC quiescence.

Through the use of the A20 conditional knockout mice (exon 2-deleted from Hammer et. al.) and

our RIP-gp DC vaccination model, we have demonstrated that A20 is required to prevent the

spontaneous maturation of DCs. Specifically, we have shown that A20 is critical for restricting

cytotoxic T cell responses and thereby preventing immune-mediated tissue destruction. Using

our model, we have shown that unstimulated A20-deficient BMDCs are able to induce CTL

function in vivo despite their reduced expression of MHCII, CD80 and CD86. Hammer et. al.,

however, observed slightly elevated expression of these molecules on splenic cDCs and pDCs

from A20 conditional knockout mice. Furthermore, this mild upregulation was found to be

independent of MyD88, suggesting that upregulation of these molecules is an intrinsic property

of A20 loss. However, our observation suggests that this is not the case.

The phenotype of DCs in conditional knockout mice may be the result of signaling

through MyD88-independent pathways (ie. not TLR, IL-1, IL-18 or IL-33). For example, A20

conditional knockout mice were shown to have greatly elevated TNFα and IL-6 serum

concentrations [188]. These cytokines are known to induce the expression of CD80 and CD86

and may therefore be driving their mild upregulation observed in A20-deficient mice [430].

However, Kool et. al. reported comparatively dramatic increases in MHCII, CD80, and CD86 in

BMDC cultures. The differences between our observations of BMDCs and those of Kool et. al.

may be due to differences in the TNFaip3 alleles employed. It could be that residual

deubiquitinases function in the BMDCs of Kool et. al may somehow potentiate the expression of

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MHCII, CD80 and CD86. Alternatively, it is possible that their reagents or culture conditions

included factors that enhance the upregulation of these molecules.

Regardless of the mechanism driving differential expression of CD80 and CD86 in our

models, our finding that CD80lo

CD86lo

DCs can induce CD8+ T cell mediated autoimmunity

suggest that neither of these molecules is essential for immunogenicity of A20-deficient DCs.

Hammer et. al. demonstrated that blockade of CD80 and CD86 using antagonistic antibodies

resulted in decreased expansion of T cells transferred into A20 conditional knockout mice.

However, we demonstrated that unstimulated A20 knockout DCs were able to induce potent

cytotoxicity and tissue immune-pathology while inducing normal T cell expansion. Together,

these findings suggest that while CD80 and CD86 on A20 knockout DCs may potentiate T cell

expansion, their upregulation is not an obligatory marker that defines a DC capable of inducing T

cell function in vivo.

We observed that unstimulated A20-deficient DCs displayed increased basal TNFα

secretion. This finding is consistent with the elevated TNFα found in A20 conditional knockout

mice, although Kool et. al. did not detect TNFα secretion by unstimulated A20 knockout

BMDCs. Dysregulated TNFα expression by A20 knockout DCs may contribute to their ability to

induce adaptive immune responses in vivo. Given the absence of NFκB proteins in unstimulated

A20 knockout DCs, the identity of the transcription factor(s) driving expression of TNFα remain

to be determined.

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A20 plays dual roles during DC maturation

In response to stimulation through PRRs, DCs undergo maturation, a process associated with

upregulated expression of MHCII, costimulatory molecules, as well as pro-inflammatory

cytokines. In addition to its role in maintaining DC quiescence, A20 plays an important role in

regulating the response to maturation stimuli. We found that A20 deficiency results in a severely

impaired ability to upregulate CD80, CD86, and MHCII in response to the TLR9 ligand CpG.

NFκB proteins drive the expression of CD80 and CD86 in DCs [380]. In particular, p50 and cRel

are thought to have crucial roles [381].The reduction of NFκB proteins expression in A20-

deficient DCs may account for their inability to upregulate CD80 and CD86. However,

expression of NFκB proteins is partially restored with CpG treatment so there may be a kinetics-

or context-dependent mechanism driving NFκB mediated expression of CD80 and CD86.

In sharp contrast to its positive role in regulating costimulatory molecules, we found that

A20 negatively regulates DC production of cytokines. A20 deficiency resulted in a greatly

enhanced ability of DCs to produce IL-12p70, IL-6, MCP-1, and IL-10 in response to CpG

stimulation. This observation is consistent with previous work that has demonstrated a role for

A20 in limiting DC cytokine production in response to LPS [187]. A recent study has

demonstrated an important role for A20 in limiting cytokine production [431]. Mice with

conditional deletion of A20 from DCs were challenged with injections of low-dose LPS.

Compared to wild type mice, conditional A20 knockout mice had greatly elevated inflammatory

cytokine production, including IL-2, IL-12 and TNFα. Furthermore, all conditional knockout

mice succumbed to this LPS-induced shock while all wild type mice survived.

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Expression of many inflammatory cytokines such as IL-12p70 and TNFα are believed to

be driven by NFκB [384, 391-393]. CpG-stimulated A20-deficient DCs express NFκB proteins

and A20 has been demonstrated to limit NFκB activity following cell stimulation. Therefore, it

may be that dysregulated NFκB signaling is promoting the exaggerated production of cytokines

following CpG stimulation. Production of pro-inflammatory cytokines and upregulation of

costimulatory molecules are often thought to be concomitant events during DC maturation. Loss

of A20, however, appears to uncouple these events. Given that both events are believed to be

driven by NFκB activity, this finding suggests that their regulation is not synonymous. Further

studies will be required to dissect out the differential requirements for the expression of

costimulatory molecules and cytokines following DC activation.

A20 and p50 maintain expression of NFκB

A20 has been characterized as a negative regulator of NFκB signaling following stimulation

through many receptors including TLRs, NLRs, TNFR, and CD40 in a variety of different cell

types [160, 163, 175, 182]. The finding that A20 deficient BMDCs have dramatically reduced

expression of NFκB proteins was therefore unexpected. This finding was also observed in

splenic DCs, demonstrating that it is not a consequence of in vitro DC generation. A20 has low

basal expression that is strongly upregulated by induced by inflammatory stimuli such as TNFα,

CD40 or TLR ligands [160, 163, 182]. The effects of A20 on NFκB have therefore been

primarily examined in the context of inflammatory signaling and not during steady-state.

Furthermore, many studies, including those that examined signaling in DCs, used the presence of

phosphorylated IκB as a surrogate marker for NFκB activation [188]. This method may not

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accurately reflect NFκB activity in the context of impaired NFκB expression. Some reports,

however, clearly demonstrate increased NFκB activity in mouse embryonic-fibroblasts (MEFs)

by EMSA [163]. Furthermore, these studies detect expression of unphosphorylated IκB in

unstimulated MEFs [163]. We additionally detected this molecular phenotype in Nfkb1-/-

DCs.

To our knowledge this phenotype has not been reported in any other Nfkb1-deficient cell type.

Together, these findings may suggest that the loss of NFκB proteins may be a phenotype unique

to DCs.

We found that while splenic A20-deficient DCs express reduced amounts of NFκB

proteins, splenic Nfkb1-deficient DCs have normal levels of NFκB subunits. This suggests that

the loss of NFκB expression may be the result of perturbations by external stimuli. A20

conditional knockout mice suffer from spontaneous inflammation including the expression of

TNFα in the serum [187]. By contrast, Nfkb1 knockout mice remain relatively healthy during

steady-state. It may be that the integration of inflammatory stimuli in A20 conditional knockout

mice is promoting DCs to adopt this molecular phenotype. BMDC cultures contain inflammatory

cytokines such as GM-CSF, IL-6, and TNFα and could therefore be promoting the adaptation of

this phenotype. Further studies will be required to carefully elucidate the requisite events

preceding the loss of NFκB expression.

A20 and the Nfkb1 proteins p50 and p105 have all been suggested to negatively regulate

NFκB signaling [102, 163, 372, 373, 432]. The substantial reduction of the various NFκB family

members observed in A20- and Nfkb1-deficient DCs may therefore be a mechanism to protect

against excessive NFκB signaling in the absence of these regulatory proteins. The best studied

mechanism of dampening NFκB activity following signaling is the negative feedback loop which

drives upregulation of IκB proteins [90]. Both A20 and Nfkb1 knockout DCs had greatly

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elevated expression of Nfkbia mRNA, which encodes for the NFκB inhibitor IκBα. This finding

further suggests that this phenotype is an attempt to limit NFκB. Notably, in spite of this

abundance of mRNA, we detected reduced expression of IκBα proteins in knockout DCs. IκB

proteins are normally found in association with NFκB dimers where they are stable are only

degraded following stimulus triggered ubiquitination [433]. By contrast, unbound IκBα is

unstable and has been demonstrated to be rapidly degraded by the proteasome through a

ubiquitin-independent process [433, 434]. Therefore, the absence of IκBα in A20- and Nfkb1-

deficient DCs is likely a consequence of the lack of NFκB dimers to bind and stabilize IκBα.

Normal termination of NFκB signaling is mediated by both the resynthesis of IκB as well

as proteasome-mediated degradation of DNA-bound NFκB dimers [133]. There have been

several reports of mechanisms by which NFκB signaling is regulated through the degradation of

NFκB proteins. The E3 ligase PDZ and LIM domain containing protein 2 (PDLIM2) has been

suggested to limit NFκB signaling through the regulation of p65 [136]. Accordingly, PDLIM2-

deficiency in a macrophage cell line resulted in the accumulation of nuclear p65 and elevated

cytokine production in response to TLR stimulation [136]. The degradation of nuclear NFκB has

also been suggested to be mediated by the EC2S complex which is composed of Elongins B and

C, Cullin-2, COMMD1, and SOCS1 [137, 435]. Here it is believed that COMMD1 facilitates the

interaction between the ECS complex and DNA-bound p65 [137]. This interaction then

facilitates the polyubiquitination of p65 by the E3 ligase SOCS1 [137]. We detected greatly

elevated Socs1 mRNA expression in A20 and Nfkb1-deficient DCs, suggesting that the ECS

complex may be contributing to the molecular phenotype of these knockout DCs. Interestingly,

SOCS1 appears to play a role in regulating DC function as loss of SOCS1 in DCs has been

suggested to dysregulated TLR signaling, promote expansion of T cells and B cells, and support

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the development of autoimmunity [436-438]. Future functional studies will be required to

confirm what role, if any, these proteins play in regulating NFκB in DCs.

Concluding Remarks

The disruption of DC homeostasis has severe consequences for the maintenance of immune

tolerance. This has been clearly demonstrated by the spontaneous autoimmunity suffered by

mice with conditional deletion of A20 from their DCs. We have demonstrated that the loss of

A20 in DCs results in spontaneous release of TNFα, a cytokine which is known to be crucial for

the immunogenicity of DCs. Previous reports suggest that upregulation of costimulatory

molecules on A20-deficient DCs drives their immunogenicity. However, we have demonstrated

that even in the absence of elevated CD80 and CD86 expression, A20 knockout DCs are able to

induce immune pathology. We have also demonstrated that the loss of quiescence in A20- and

Nfkb1-deficient DCs is accompanied by the dramatic loss of NFκB transcription factor

expression, a phenomenon which is mediated through protein degradation. Finally, we have

identified a candidate E3 ubiquitin ligase, SOCS1, which is upregulated in both knockout DCs

and may therefore be driving the loss of NFκB.

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Chapter VI

Discussion

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The regulation of homeostatic signals

In the absence of an immunological challenge, the abundance, distribution, and functional

differentiation of immune cells is maintained in a steady-state. The preservation of this

homeostasis is dependent upon numerous signals. The survival of peripheral T cells, for

example, is reliant upon signals from both self-peptide MHC complexes and IL-2Rγc-dependent

cytokines such as IL-7 [439]. Likewise, the cytokine Flt3L is required to sustain the continued

replenishment of DCs in lymphoid organs [8]. In addition to these endogenous signals, it is now

apparent that microbiota-derived signals also contribute to immune homeostasis. The recognition

of commensal bacteria through various PRRs is required to maintain the homeostasis of the

intestinal epithelial cells and intraepithelial lymphocytes [440, 441]. The effects of these

microbiota-derived signals may extend beyond the gut; peripheral lymphocytes may also be

affected by perturbations to the intestinal flora [442].

The proper integration of these environmental cues is required to sustain homeostasis.

Dysregulation of signaling from these cues may result in perturbed homeostasis. For example,

expression of the IL-7Rα chain is controlled by the transcription factor Forkhead box protein o1

(Foxo1). Deficiency in Foxo1 results in impaired IL-7Rα expression and, consequently,

compromised survival of naïve T cells [443]. Furthermore, Foxo1 deficiency leads to an

expansion of memory-phenotype T cells, likely due to the homeostatic proliferation of T cells

required to fill the T cell niche following the death of naïve cells. Although IL-4 also signals

through IL-2Rγc and may promote T cell survival [439], it is not believed to contribute to the

homeostasis of naïve T cells [444]. However, it is known that IL-4 is produced during

homeostasis and plays a role in the maintenance of type 2 macrophage [445, 446]. We

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demonstrated in Chapter III that Shp1-deficient T cells are hypersensitive to homeostatic levels

of IL-4. Therefore, the regulation of Stat6 by Shp1 may therefore be viewed as a mechanism

which prevents T cells from erroneously responding to a homeostatic signal.

Shp1 has been found to play an analogous role in DC biology. DC-specific ablation of

Shp1 expression results in spontaneous DC activation, the accumulation of activated T cells and

B cells, and the presence of anti-nuclear antibodies [276]. It was demonstrated that these findings

were a consequence of dysregulated MyD88-dependent signaling. Additionally, Shp1-deficient

DCs were found to be hypersensitive to stimulation through TLR agonists. These findings are

analogous to those made with mice harboring A20-deficient DCs [187]. Like Shp1-deficiency,

A20-deficiency in DCs results in TLR hypersensitivity and a spontaneous MyD88-dependent

expansion of activated T cells. Together, these data suggest that A20 and Shp1 prevent the

activation of DCs and lymphocytes in these mice by limiting signaling from TLR agonists

present during homeostasis. However, the identities of these homeostatic TLR agonists, whether

microbial or endogenous in origin, remain unknown.

Further evidence for the presence of homeostatic TLR agonists which may interact with

DCs comes from experiments with mice engineered to overexpress TLR7, which may recognize

both host and viral nucleic acids [447]. These mice develop spontaneous DC activation,

expansion of lymphoid and myeloid compartments, and autoantibody production.

Furthermore, adherent localization of TLR9 to the cell surface resulted in DC activation and

inflammatory disease, further suggesting that endogenous TLR agonists may regulate DC

biology [448]. DC quiescence is therefore maintained by various mechanisms, including the

negative regulatory molecules Shp1 and A20, which limit the ability of immature DCs to

respond to TLR signals.

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Active regulation of immune cell quiescence

In homeostasis, immune cells primarily exist in their naïve or immature states. Upon infection,

however, the introduction of new exogenous signals in the form of PRR agonists and

pathogen-derived antigens promotes the activation of immune cells and the induction of

immunity. These observations led to the idea that immune cells are quiescent by default and

require these exogenous signals to induce their activation. However, this understanding has been

challenged by observations, including those displayed in Chapters III, IV, and V, which

suggest that intrinsic regulatory factors are required to maintain quiescence.

A naïve T cell is characterized not only by its lack of proliferation, by also by its

expression of various molecules which control its survival and trafficking. This naïve state has

been demonstrated to be under the control of various factors. Deficiency in these molecules,

including Foxo1 [443], Foxp1 [449], Kruppel-like factor 2 (Klf2) [450, 451], Schlafen-2 (Slfn2)

[452], and Tuberous sclerosis complex 1 (Tsc1) [453], results in a loss of naïve T cells and a

concomitant increase in memory-phenotype T cells. Our findings in Chapter III demonstrate

that Shp1 is also a factor required to maintain T cells in a quiescent state. Some of these factors,

including Foxo1 and Foxp1, appear to function through regulating sensitivity to homeostatic

signals such as IL-7. Others, including Tsc1 appear to have a more direct impact on the survival

of naïve cells. T cell quiescence is therefore more than a passive state and is actively maintained

by various regulatory molecules.

DC immaturity has also been widely characterized as a default state. Furthermore, it has

been suggested that exogenous stimuli, through their activation of PRR molecules such as TLRs,

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are essential to induce the functional maturation of DCs. However, new data, including those

presented in Chapters IV and V, suggest that DC quiescence is actively regulated. Furthermore,

some of these regulatory mechanisms act not through regulation of exogenous TLR stimuli, but

through intrinsic maintenance of DC immaturity.

The generation of DC-specific A20 knockout mice identified A20 as a regulatory

molecule required to maintain DC quiescence [187, 188]. While the phenotype of these mice was

attenuated by loss of MyD88, A20- MyD88-double mutant mice still contained elevated levels of

activated T cells. This finding suggests that the role of A20 in DCs may extend beyond the

regulation of MyD88-dependent receptors such as the TLRs. Indeed, our data from Chapter V

demonstrated that A20-deficient DCs have an enhanced ability to generate CD8+ T cell

responses even in the absence of TLR agonists. Our laboratory identified Nfkb1 as an additional

DC quiescence factor [374]. Furthmore, in Chapter IV, we identified p50 as the Nfkb1 gene

product which is essential for preventing spontaneous functional DC maturation. As with A20-

deficient DCs, Nfkb1-deficient DCs are able to generate T cell responses independently of TLR

activation. However, unlike the loss of A20, Nfkb1-deficiency does not appear to enhance

sensitivity to TLR stimulation.

Destabilization of the immature DC state

The functional properties of A20- and Nfkb1-deficient DCs parallels that of a conventionally

activated DC; the loss of these DC quiescence factors and TLR stimulation both result in the

ability to promote T cell activation and consequently induce immune-pathology. However, other

phenotypic qualities of these populations are distinct. TLR stimulation results in robust

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upregulation of CD80, CD86, and MHCII. By contrast, A20- and Nfkb1-deficient DCs have

similar or slightly reduced expression of these molecules in comparison to immature DCs.

Furthermore, TLR activation of DCs results in ample production of inflammatory cytokines such

as TNFα, IL-12, and IL-6. A20- and Nfkb1 DCs spontaneously produce TNFα, albeit at levels

less than TLR-stimulated DC. Therefore, the functional and phenotypic properties of A20- and

Nfkb1-deficient DCs resemble neither those of immature DC nor those of conventionally

matured DCs. Furthermore, we demonstrated in Chapter V that dramatic alterations to the

regulation of the NFκB signaling axis are present in these DCs. We have labeled these DCs as

“destabilized”, in reference to both their deviation from immaturity and loss of stable NFκB

expression (Figure VI-1). The precise mechanism by which destabilized DCs are able to induce

T cell immunity is still not entirely clear, although an important role for TNFα has been

established in the case of Nfkb1-deficient DCs.

The immature DC state is therefore actively regulated by negative regulatory factors in

two conceptually distinct manners. These factors may negatively regulate TLR signaling, as is

the case for Shp1 and A20. Alternatively, or additionally, these factors may be required to

maintain the immature state even in the absence of exogenous stimulation, as is the case for

Nfkb1 and A20. These factors therefore prevent the adaptation of a destabilized phenotype,

maintaining the functional immaturity of the DC.

While our identification of this phenotype arose from the examination of genetically

altered cells, DC destabilization could also occur in conventional DCs. For example, it could be

that specific genetic backgrounds may predispose DCs to destabilization. For example, it may be

that DCs harboring the -863A TNFα promoter mutation, which prevents p50 homodimer-

mediated inhibition of TNFα, could display characteristics of destabilized DCs. The

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identification of destabilized DCs may be difficult given that the discriminable surface

phenotype of immature and destabilized DCs. Therefore, identifying the conditions which may

promote the destabilization of an immature DC will be crucial for extending our understanding

of this phenotype. Splenic Nfkb1 DCs do not display the loss of NFκB proteins as destabilized

DC populations do. It may be that specific conditions are required to destabilize Nfkb1 DCs.

Therefore, studying the transition of Nfkb1 DCs from an immature to destabilized phenotype

may provide essential clues to understanding this phenomenon.

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Figure VI-1. A destabilized DC phenotype induces T cell immunity. Conventional DC

maturation is induced through activation of TLRs, resulting in activation of NFκB, the

expression of inflammatory cytokines, MHC molecules, and costimulatory molecules, and the

induction of T-cell immunity. Dysregulation of NFκB, such as through the loss of A20 or Nfkb1

results in a destabilized DC phenotype characterized by loss of NFκB expression, increased

expression of TNFα, and the ability to induce T cell responses in vivo.

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The ablation of NF𝜅B expression in destabilized DCs

The process of conventional DC maturation is associated with the activation of NFκB

transcription factors. Therefore, perhaps the most unexpected observation of destabilized DCs

was that the expression of NFκB proteins in these DCs is dramatically reduced. This finding

illustrates the distinct molecular phenotypes of activated and destabilized DCs. Furthermore, it

spurs several interesting questions regarding the biology of NFκB in DCs. Firstly, what

mechanism is driving the loss of NFκB expression? The degradation of NFκB proteins has been

previously described, and has been suggested to be mediated by several different E3 ubiquitin

ligases. However, these mechanisms have only been demonstrated to target DNA-bound NFκB

following NFκB activation. In combination with the described regulatory roles for A20 and

Nfkb1 in NFκB signaling, these reports may suggest that the degradation of NFκB proteins in

destabilized DCs may be a response to their aberrant DNA binding. An examination of the

localization of NFκB proteins following the blockade of their degradation may provide evidence

to support or refute this hypothesis. Alternatively, the degradative mechanism active in

destabilized DCs may be distinct from those previously described and could be targeting

cytoplasmic NFκB. Notably, the expression of NFκB proteins is restored following treatment

with a TLR agonist. This finding suggests that TLR stimulation regulates the degradative

mechanism. Therefore, it may be that this observation reflects a mechanism which is able to

discriminate between dysregulated NFκB signaling and genuine NFκB-activating stimuli.

Furthermore, what consequence does the loss of NFκB expression have for DC function?

Are the degradation of NFκB proteins and the acquisition of immunostimulatory properties two

distinct repercussions of dysregulated NFκB signaling? Or does the loss of NFκB actually

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potentiate the immunogenicity of destabilized DCs? If the latter is true, it points to a role for

NFκB proteins in maintaining functional immaturity. It could be that this is accomplished

through the activities of p50 homodimers, which may prevent the transcription of TNFα or other

pro-inflammatory molecules by other transcription factors. Alternatively, NFκB dimers may be

required to drive the transcription of other genes which maintain DCs in an immature state. This

novel observation may therefore provide the basis for exploring new mechanisms regulating both

NFκB and DC quiessence.

Co-stimulation of T cell responses

Members of the B7 family have been frequently characterized as important co-stimulatory

molecules driving T cell activation. While these molecules are upregulated on activated DCs and

they can promote T cell activation, their expression does not necessarily coincide with the

generation of T cell responses.

In Chapters IV and V, we reported that destabilized DC populations are able to induce

T cell-mediated immune pathology in spite of their low expression of the B7 molecules CD80

and CD86. This finding is consistent with a report of Heat shock protein 70 (Hsp70) activated

DCs which were shown to be functional mature in the absence of B7 upregulation [454]. In vivo

administration of Hsp70 along with peptide was found to enhance the immunogenicity of DCs

leading to the induction of a robust cytotoxic T cell response. While there was no concurrent

upregulation of CD80, CD86, or MHCII, Hsp70 did induce the production of IL-12 in DCs.

Furthermore, while T cell responses against some pathogens are CD28-dependent, others such as

those against LCMV and murine gamma herpesvirus are independent of B7-CD28 interactions

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157

[209]. This observation further demonstrates that costimulation through CD28 is not an absolute

requirement for the generation of T cell immunity.

The induction of diabetes in RIP-gp mice by CpG-stimulated DCs, however, is dependent

upon CD28 [455]. How is it then, that A20- and Nfkb1-deficient DCs are able to induce diabetes

without upregulating CD80 and CD86? One possibility is that the basal levels of CD80 and

CD86 expression found on unstimulated DCs are sufficient for the induction of diabetes. This

would suggest that CD80 and CD86 upregulation on wildtype DCs is not the dominant

mechanism by which TLR stimulation induces their functional maturation. Alternatively, the

destabilized A20 and Nfkb1 knockout DCs may be promoting T cell activation in a CD28-

independent manner. If this was found to be true, it would reinforce the distinct phenotypes of

conventionally activated and destabilized DCs.

Several studies have reported DC populations that are phenotypically mature, but do not

induce immunity [49-51]. One common property of these mature DCs is that they do not produce

inflammatory cytokines, suggesting that upregulation of costimulatory molecules in the absence

of inflammatory cytokines is insufficient to promote T cell activation. Furthermore, in some

settings, phenotypically mature DCs have been suggested to promote immune tolerance [52].

One study demonstrated that TNF-treated DCs, which upregulated expression of CD80 and

CD86, were able to induce tolerance in the experimental autoimmune encephalomyelitis model

of multiple sclerosis. This was a feat that both immature and TLR-matured DCs could not

perform. In summary, while conventional dendritic cell maturation induces expression of B7

family costimulatory molecules, their upregulation is neither necessary nor sufficient to support

the induction of T cell immunity.

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Concluding Remarks

The immune system is unrivaled in its ability to transition from a state of apparent

quiescence to one of robust activation. This phenomenon is a manifestation of the abundant

molecular check and balances which govern the proliferation and differentiation of immune

cells. Following the resolution of a response, immune cells return to a state of quiescence and are

once again seemingly dormant.

This stillness, however, is superficial. The quiescent immune system, like one mounting a

response, is prudently regulating its cells and molecules. Immune cells are often thought to be

quiescent by default, requiring exogenous signals to drive their activation. However, mounting

evidence presented here and elsewhere has demonstrated that key regulatory molecules are

required to prevent their spontaneous activation. These molecules may therefore be of crucial

importance to sterile immune responses that lack conventional activation stimuli. Consequently,

a thorough understanding of these molecules may be essential for the prediction and

manipulation of immune responses in the settings of autoimmunity or malignancy.

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Copyright Information

Chapter III was modified from a publication:

Johnson DJ, Pao LI, Dhanji S, Murakami K, Ohashi PS, Neel BG. 2013. Shp1 regulates T cell

homeostasis by limiting IL-4 signals. J Exp Med 210: 1419-31