The Maintenance of Immune Homeostasis and Quiescence by ...
Transcript of 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
2
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,
14
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.
15
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
16
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].
17
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
18
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
19
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.
20
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.
21
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
22
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.
23
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
24
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,
25
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
26
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.
27
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
28
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
29
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
30
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
31
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
32
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
33
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
34
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
35
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].
36
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.
37
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)
38
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.
39
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.
40
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)
41
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
42
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
43
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
44
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.
45
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
46
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
47
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
48
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
49
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.
50
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
51
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].
52
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].
53
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
54
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
55
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].
56
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
57
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
58
[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.
59
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
60
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.
62
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
63
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
64
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.
65
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).
66
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).
67
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.
68
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
69
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.
70
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].
71
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.
72
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.
73
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
74
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
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).
76
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.
77
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.
78
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.
79
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
80
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
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
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.
83
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
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.
85
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
86
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
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
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.
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
90
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.
92
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.
93
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
94
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
95
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.
96
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
98
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.
100
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.
101
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
102
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.
103
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.
105
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.
106
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
107
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.
108
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.
109
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
110
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
111
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.
112
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.
113
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
115
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.
116
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,
117
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
118
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
119
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].
122
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.
123
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.
125
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.
126
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
127
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.
128
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
129
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
130
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.
131
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.
132
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).
133
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
134
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.
135
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
136
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
137
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.
138
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
139
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
140
[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
141
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.
143
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
144
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
145
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
146
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.
148
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
149
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.
150
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,
151
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
152
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
153
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.
154
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.
155
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
156
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
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.
158
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.
159
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