regulation of anti-viral immunity by dendritic cells and ... · 1.5.2 armstrong vs. clone 13:...
TRANSCRIPT
Regulation of anti-viral immunity
by dendritic cells and natural killer cells
by
Achire Nathalia Mbanwi
A thesis submitted in conformity with the requirements
for the degree of Doctor of Philosophy
Department of Immunology
University of Toronto
© Copyright by Achire Nathalia Mbanwi 2015
ii
Regulation of anti-viral immunity by dendritic cells and natural killer cells
Achire Nathalia Mbanwi
Doctor of Philosophy
Department of Immunology
University of Toronto
2015
Abstract
4-1BB and its ligand, 4-1BBL, are costimulatory members of the tumor necrosis factor receptor
(TNFR)/TNF family. The 4-1BB/4-1BBL pathway has been shown to potently enhance T cell
responses in models of viral infection and cancer, thereby identifying this pathway as a target for
immunotherapy. In addition to T cells, 4-1BB is expressed on dendritic cells (DCs), which also
express 4-1BBL. The precise nature of 4-1BB/4-1BBL expression on DCs is not known and the
endogenous function of 4-1BB/4-1BBL on this cell type has remained largely elusive. Here, I
show that LPS-activated bone marrow-derived DCs express both 4-1BB and 4-1BBL, and that
these molecules constitutively interact on DCs. Survival, upregulation of key costimulatory
molecules, cytokine/chemokine production, antigen presentation, and the ability of DCs to
generate primary influenza-specific CD8 T cell responses were normal in the absence of 4-1BB
and/or 4-1BBL on DCs. Thus, the biological implications of 4-1BB/4-1BBL interactions on DCs
remain to be elucidated.
It was previously demonstrated in the Watts lab that the 4-1BB pathway on T cells becomes
desensitized during persistent infection with lymphocytic choriomeningitis virus (LCMV) clone
13 through loss of the signaling adapter TRAF1. In the course of these studies, it was found that
iii
2 months following LCMV clone 13 infection, a time point at which virus is largely cleared from
the spleen and other organs, C57BL/6 mice showed dramatic splenic atrophy. Here, I show that
this atrophy persists for up to 10 months following initial infection and that it does not occur
with the acutely infecting strain, LCMV Armstrong. I go on to show that splenic atrophy is
mediated in part by natural killer (NK) cells, and implicate NKG2D-MULT1 interactions as
contributing to late-stage B cell depletion. Compared to isotype control antibody treatment, type
I interferon receptor 1 (IFNAR1) blocking antibody treatment at the onset of splenic atrophy
increased the proportion of NK cells in the spleen, and was correlated with exacerbated splenic
atrophy. Lastly, splenic atrophy following LCMV clone 13 infection was associated with delayed
clearance of a bacterial pathogen. These findings implicate NK cells in the pathophysiological
mechanisms underlying splenic atrophy following viral infection.
iv
Acknowledgments
First and foremost, I would like to thank my supervisor, Dr. Tania Watts, for the opportunity to
take on graduate studies in her lab. Over the years, she has been a source of support and
encouragement, and her enthusiasm for science kept me motivated and excited about the work. I
thank her for providing her decades of experience in asking the right questions, designing
experiments, and scientific writing, but also for granting me some freedom in pursuing my own
ideas. I am certainly grateful that I was able to attend 4 conferences throughout my graduate
studies, especially the 100th
annual American Association of Immunologists conference in
Honolulu, Hawaii.
I thank my committee members, Dr. Dana Philpott and Dr. Juan Carlos Zúñiga-Pflücker, for
their thought-provoking questions and ideas during committee meetings, and for being kind and
approachable.
I would also like to recognize the past and present members of the Watts lab for their technical
assistance and expertise, but also for providing encouragement and emotional support when
things went wrong.
Last, but not least, I would like to express my deepest gratitude to my family and friends for their
incredible love and support throughout my graduate studies. When experiments worked, they
were there for the good times. When experiments failed, they were there for the bad times. I
thank them most importantly for always believing in me and pushing me out of my comfort
zone, and for providing that support network that no one can do without. Special thanks to my
parents, Eta and Bershu, my aunt Bertha, and my two little brothers, Sikem and Matthew.
v
Table of Contents
Abstract ii
Acknowledgments iv
Table of Contents v
List of Tables xi
List of Figures xii
List of Publications xiv
Abbreviations xv
Chapter 1: Introduction 1
1.1 Overview 2
1.2 The innate immune response in anti-viral immunity 2
1.2.1 Dendritic cells 3
DC development and homeostasis 4
DC subsets 4
Functions of DCs 5
1.2.2 DCs in anti-viral immunity 6
1.2.3 Therapeutic implications of DCs: DC vaccination 7
1.2.4 Natural killer cells 8
NK cell development and homeostasis 8
NK cell subsets 9
vi
NK cell receptors and ligands 10
Functions of NK cells 14
1.2.5 NK cells in anti-viral immunity 15
1.3 The spleen as a secondary lymphoid organ involved in anti-viral immunity 16
1.3.1 Development of the spleen 16
1.3.2 Structure and function of the spleen 18
1.4 Splenic atrophy due to viral infection and other causes 20
1.4.1 Splenic atrophy in sickle cell anemia and autoimmunity 21
1.4.2 Splenic atrophy due to viral infection: what are the mechanisms? 21
1.4.3 Compromised immunity due to complications associated with the spleen 23
1.5 Murine models of viral infection: LCMV 23
1.5.1 LCMV virus 24
1.5.2 Armstrong vs. clone 13: mechanisms underlying clearance vs. persistence 24
1.5.3 The innate immune response to LCMV infection 27
1.5.4 The role of NK cells during LCMV infection 28
1.5.5 Type I Interferons during LCMV infection 32
1.5.6 Splenic architecture and remodeling following LCMV infection 34
1.5.7 Resolution of inflammation following LCMV infection 36
1.6 Murine models of viral infection: influenza 37
1.6.1 Influenza virus 37
1.6.2 Innate immune responses 38
vii
1.6.3 Adaptive immune responses 39
1.7 TNF/TNFR family 39
1.7.1 Overview 40
1.7.2 4-1BB/4-1BBL 40
1.7.3 Role of 4-1BB/4-1BBL on T cells 41
1.7.4 Role of 4-1BB/4-1BBL on non-T cells 43
1.7.5 Role of 4-1BB/4-1BBL on DCs 43
1.7.6 4-1BB signaling 44
1.8 Thesis Rationale 47
Chapter 2: 4-1BB and 4-1BBL constitutively interact on LPS-activated dendritic cells
but dendritic cell-intrinsic 4-1BB/4-1BBL interactions are dispensable for
cytokine production and anti-viral CD8 T cell priming 48
2.1 Abstract 49
2.2 Introduction 49
2.3 Materials and Methods 50
2.3.1 Mice 50
2.3.2 DC cultures and surface markers 51
2.3.3 Blocking studies 51
2.3.4 DC survival assay 52
2.3.5 Cytokine production 52
2.3.6 In vitro antigen uptake and presentation 52
2.3.7 DC vaccination 53
viii
2.3.8 Flow cytometry 54
2.3.9 Lentiviral transductions and confocal microscopy 54
2.3.10 Statistical Analysis 54
2.4 Results 54
2.4.1 Chacterization of DCs lacking 4-1BB and 4-1BBL 54
2.4.2 4-1BBL is not readily detectable in the presence of 4-1BB on LPS-activated
DCs 56
2.4.3 The surface expression of 4-1BBL can be regulated extrinsically 56
2.4.4 Evidence of constitutive interaction between 4-1BB and 4-1BBL on DCs 59
2.4.5 Indistinguishable in vitro survival of WT and DKO DCs 63
2.4.6 4-1BB and/or 4-1BBL do not impact the level of the cytokines IL-2, IL-6,
IL-12, IL-18, and TNF, nor the chemokines MCP-1 and RANTES in LPS-
activated DCs 63
2.4.7 4-1BB and 4-1BBL are dispensable for antigen presentation by DCs
in vitro 63
2.4.8 DC-intrinsic 4-1BB and 4-1BBL are largely dispensable for T cell priming
against a viral antigen in vivo 66
2.5 Discussion 72
Chapter 3: Natural killer cells contribute to splenic atrophy observed months following
LCMV clone 13 infection 76
3.1 Abstract 77
3.2 Introduction 77
3.3 Materials and Methods 79
ix
3.3.1 Mice and infections 79
3.3.2 In vivo antibody treatments 79
3.3.3 Flow cytometry and antibodies 80
3.3.4 Immunofluorescence microscopy 80
3.3.5 Statistical analysis 81
3.4 Results 81
3.4.1 Persistent splenic atrophy following infection with LCMV clone 13 81
3.4.2 Atrophic spleens are lymphopenic, but show T cell/B cell segregation within the
white pulp 84
3.4.3 NK cells contribute to splenic atrophy following LCMV clone 13
infection 86
3.4.4 IFNAR1 blocking antibody treatment increases the proportion of NK cells, and
exacerbates splenic atrophy 89
3.4.5 Splenic atrophy delays clearance of a bacterial pathogen 92
3.4.6 The proportion of LTi is similar between aged naive and LCMV-
infected mice 94
3.5 Discussion 96
Chapter 4: Discussion and Future Directions 101
4.1 Overview 102
4.2 Role of 4-1BB/4-1BBL on DCs: What unique transcripts are induced by 4-1BB/
4-1BBL on DCs and does this impact secondary immune responses? 102
4.3 4-1BB signaling in DCs 104
4.4 Potential bidirectional signaling by 4-1BB and 4-1BBL on DCs 105
x
4.5 Splenic atrophy following LCMV clone 13 infection 106
4.6 Can the spleen regenerate following splenic atrophy? 109
4.7 Conclusion 111
Chapter 5: References 113
xi
List of Tables
Chapter 1
Table 1.1 Major mouse and human NK cell receptors 11
xii
List of Figures
Chapter 1
Figure 1.1: Structure of the spleen 19
Figure 1.2: Signaling downstream of 4-1BB on T cells 46
Chapter 2
Figure 2.1: LPS-induced upregulation of MHC-I, CD80, CD86, and CD40 is normal
in the absence of 4-1BB and 4-1BBL 55
Figure 2.2: 4-1BBL is not readily detectable in the presence of 4-1BB on LPS-activated
DCs 57
Figure 2.3: The surface expression of 4-1BBL can be regulated extrinsically 58
Figure 2.4: Citrine expression is detectable on the surface of 4-1BBL-citrine-transduced
WT and 4-1BB-/-
DCs 60
Figure 2.5: Evidence of constitutive interaction between 4-1BB and 4-1BBL on DCs 61
Figure 2.6: 4-1BB and 4-1BBL are dispensable for DC survival in vitro 64
Figure 2.7: 4-1BB and/or 4-1BBL are not required for IL-2, IL-6, IL-12, IL-18, MCP-1,
RANTES, and TNF production by DCs 65
Figure 2.8: 4-1BB and 4-1BBL are dispensable for DC antigen presentation in vitro 67
Figure 2.9: DC-intrinsic 4-1BB and 4-1BBL are largely dispensable for T cell priming
against a viral antigen in vivo 69
Chapter 3
Figure 3.1: Persistent splenic atrophy following infection with LCMV clone 13 82
Figure 3.2: Atrophic spleens are lymphopenic, but show T cell/B cell segregation within the
white pulp 85
xiii
Figure 3.3: NK cells contribute to splenic atrophy 87
Figure 3.4: IFNAR1 blocking antibody treatment increases the proportion of NK cells, and
exacerbates splenic atrophy 90
Figure 3.5: Splenic atrophy delays clearance of a bacterial pathogen 93
Figure 3.6: The proportion of LTi is similar between aged naive and LCMV-infected
mice 95
Chapter 4
Chapter 4.1: 4-1BB/4-1BBL constitutive interaction on bone marrow derived DCs
in vitro 103
Chapter 4.2: NK cells contribute to splenic atrophy following LCMV clone 13
infection 107
xiv
List of Publications
1. Mbanwi AN, Lin GH, Sabbagh L, Watts TH. 4-1BB and 4-1BBL constitutively interact
on LPS-activated dendritic cells but dendritic cell-intrinsic 4-1BB/4-1BBL interactions
are dispensable for cytokine production and anti-viral CD8 T cell priming.
Manuscript in preparation.
2. Mbanwi AN, Wang C, Geddes K, Philpott DJ, Watts TH. Natural killer cells contribute
to splenic atrophy observed months following LCMV clone 13 infection.
Manuscript in preparation.
3. Mbanwi AN, Watts TH. 2014. Costimulatory TNFR family members in control of viral
infection: outstanding questions. Semin Immunol 26: 210-9
4. Lin GH, Edele F, Mbanwi AN, Wortzman ME, Snell LM, Vidric M, Roth K, Hauser AE,
Watts TH. 2012. Contribution of 4-1BBL on radioresistant cells in providing survival
signals through 4-1BB expressed on CD8(+) memory T cells in the bone marrow. Eur J
Immunol 42: 2861-74
xv
Abbreviations
(i)NKT – (invariant) natural killer T cell
(i)Treg – (inducible) regulatory T cell
4-1BBL – ligand of 4-1BB
7-AAD – 7-aminoactinomycin D
Ad - adenovirus
ADCC – antibody-dependent cellular cytotoxicity
AICD – activation-induced cell death
AIDS – acquired immunodeficiency syndrome
AP – alkaline phosphatase
APC – allophycocyanin
APC – antigen presenting cell
ARM – LCMV Armstrong
BAFF – B cell-activating factor of the tumor necrosis factor family
Bcl – B cell lymphoma
BIR – baculovirus inhibitor of apoptosis protein repeat
BM – bone marrow
BrdU – 5’-bromo-2’-deoxyuridine
CCL – CC chemokine ligand
CCR – CC chemokine receptor
CD – cluster of differentiation
xvi
cDC – conventional dendritic cell
cDNA – complementary DNA
CDP – committed dendritic cell progenitor
CDR – complementarity determining region
CFSE – carboxyfluoroscein succinimidyl ester
CFU – colony forming unit
cIAP – cellular inhibitor of apoptosis
CKI – casein kinase I
Cl13 – LCMV clone 13
CMV – cytomegalovirus
CT – computed tomography
CTL – cytotoxic T lymphocyte
CXCL – chemokine (C-X-C) motif ligand
CXCR – chemokine (C-X-C) motif receptor
DAPI – 4’,6-diamino-2-phenylindole
DC – dendritic cell
DC-SIGN – dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin
DD – death domain
DKO – double knockout
DLN – draining lymph node
DNA – deoxyribonucleic acid
xvii
dpi – days post-infection
dsRNA – double-stranded ribonucleic acid
ELISA – enzyme-linked immunosorbent assay
ERK – extracellular signal-regulated kinase
ES – embryonic stem
Fc – fragment crystallizable
FDC – follicular dendritic cell
FITC – fluorescein isothiocyanate
FLT3(L) – FMS-like tyrosine kinase 3(ligand)
FMO – fluorescence minus one
FOXO – forkhead transcription factor, class O
FRC – fibroblastic reticular cell
FSC – forward scatter
FV – Friend virus
GALT – gut-associated lymphoid tissue
GFP – green fluorescent protein
GITR – glucocorticoid induced tumor necrosis factor receptor related protein
GM-CSF – granulocyte-macrophage colony-stimulating factor
GP – glycoprotein
GPPS – glutamine, sodium pyruvate, penicillin streptomycin
HA – hemagglutinin
xviii
HBV – hepatitis B virus
HCV – hepatitis C virus
HEK – human embryonic kidney
HIV – human immunodeficiency virus
HLA – human leukocyte antigen
Hox11 – homeobox 11
HSC – hematopoietic stem cell
HSV1 – herpes simplex virus 1
i.p. – intraperitoneal
i.v. – intravenous
ICAM – intercellular adhesion molecule
IFITM – interferon-induced transmembrane protein
IFN – interferon
IFNAR1 – type I interferon receptor 1
Ig – immunoglobulin
IκBα – inhibitor of NF-κB
IKK – IκB kinase
IL – interleukin
IRF – interferon-regulatory factor
ISG – interferon-stimulated gene
ITAM – immunoreceptor tyrosine-based activating motif
xix
ITIM – immunoreceptor tyrosine-based inhibitory motif
JNK – c-Jun N-terminal kinase
KIR - killer cell immunoglobulin-like receptor
KLRG1 – killer cell lectin-like receptor G1
LCMV – lymphocytic choriomeningitis virus
LN – lymph node
LPS – lipopolysaccharide
LSECtin – lymph node sinusoidal endothelial calcium-dependent lectin
LSP-1 – lymphocyte-specific protein 1
LT – lymphotoxin
LTi – lymphoid tissue inducer cells
LTo – lymphoid tissue organizer cell
LTβR – lymphotoxin-beta-receptor
MAPK – mitogen activated protein kinase
MARCH – membrane-associated RING-CH
MCMV – murine cytomegalovirus
MCP-1 – monocyte chemoattractant protein-1
MDP – monocyte and dendritic cell progenitor
MDSC – myeloid-derived suppressor cell
MFI – mean fluorescence intensity
MHC – major histocompatibility complex
xx
MHV68 – murine gammaherpes virus 68
MKK – MAP kinase kinase
MKKK – MAP kinase kinase kinase
MLN – mediastinal lymph node
MOI – multiplicity of infection
MULT1 – murine UL16-binding protein-like transcript 1
NA – neuraminidase
NCR1 – natural cytotoxicity receptor 1
NEAA – non-essential amino acids
NFAT – nuclear factor of activated T cells
NF-κB – nuclear factor κB
NIK – NF-κB-inducing kinase
NK – natural killer
NKG2D – natural-killer group 2D
NLR - nucleotide oligomerization domain-like receptor
NP – nucleoprotein
OPT - optical projection tomography
OVA – ovalbumin
PAMP – pathogen-associated molecular pattern
PBS – phosphate buffered saline
PD – programmed death
xxi
pDC – plasmacytoid dendritic cell
PE – phycoerythrin
PFU – plaque forming unit
pi – post-infection
PI3K – phosphatidylinositol-3 kinase
PLN – peripheral lymph node
PP –Peyer’s patch
PRR – pattern recognition receptor
RAE1 – retinoic acid early inducible gene 1
RAG – recombination-activating gene
RALDH – retinoic acid-producing retinaldehyde dehydrogenase
RANK – receptor activator of NF-κB
RANTES – regulated on activation, normal T cell expressed and secreted
RBC – red blood cell
RCMV – rat CMV
RCTL – RMCV C-type lectin-like
RDC – respiratory dendritic cell
RING – really interesting new gene
RIP1 – receptor interacting protein 1
RLR - retinoic acid-inducible gene I-like receptor
RORγt – retinoic acid receptor-related orphan receptor gamma t
xxii
SA – streptavidin
SAP – signaling lymphocytic activation molecule-associated protein
SARS – severe acute respiratory syndrome
SH2 – Src homology 2
SHIP – SH2-containing inositol polyphosphate 5-phosphatase
SHP – Src homology 2-containing tyrosine phosphatase
SIINFEKL – OVA257-264
SIV – simian immunodeficiency virus
SLE – systemic lupus erythematosus
SLO – secondary lymphoid organ
SMP – splanchnic mesodermal plate
SSC – side scatter
ssRNA – single-stranded ribonucleic acid
STAT – signal transducer and activator of transcription
TAM – tyro3 axl mer
TBEV - Tick-borne encephalitis virus
TBS(T) – Tris-buffered saline (Tween-20)
TCF-1 – T cell factor 1
TCR – T cell receptor
Tfh – T follicular helper cell
TGFβ – transforming growth factor β
xxiii
Th – T helper
TIRAP - Toll/IL-1R domain-containing adapter protein
TLR – toll-like receptor
TNF – tumor necrosis factor
TNFR – tumor necrosis factor receptor
TRAF – tumor necrosis factor receptor associated factor
TRAIL – tumor necrosis factor-related apoptosis-inducing ligand
TRAM - toll/IL-1R domain-containing adapter-inducing interferon-β -related adapter molecule
TRIF - toll/IL-1R domain-containing adapter-inducing interferon-β
UV – ultraviolet
VEGF – vascular endothelial growth factor
VCAM – vascular adhesion molecule
VSV – vesicular stomatitis virus
VV – vaccinia virus
WT – wild-type
α-DG – alpha-dystroglycan
1
Chapter 1
Introduction
2
1.1 Overview
The innate immune system represents the first line of defense against the microbial world.
Among innate immune cells, both DCs and NK cells have emerged as key players in
orchestrating the immune response against viral pathogens. In this thesis introduction, I will first
discuss the general biology of these cells, and then highlight some of their roles in anti-viral
immunity. The spleen is the largest secondary lymphoid organ in the body, and the work
presented in this thesis involves anti-viral immune responses generated in this organ. I will
therefore discuss the development and structure of the spleen, as well as its role in immunity. In
chapter 3 of this thesis, I show that persistent infection of mice with LCMV clone 13 results in
sustained splenic atrophy that is mediated in part by NK cells. Splenic atrophy is commonly
associated with autoimmune disease, but has been shown to occur following viral infection in
some models. I will discuss these models and postulate on the mechanisms controlling spleen
size. I will then introduce the models of viral infection used in this thesis, namely LCMV and
influenza infection of mice. Members of the TNFR family, in particular 4-1BB and its ligand 4-
1BBL, have been shown to modulate various aspects of T cell biology following influenza
infection. However, 4-1BB is more widely expressed on cells of the immune system, and in
chapter 2, I investigate 4-1BB and 4-1BBL expression and function on DCs. Therefore, I will
end the thesis introduction with a discussion of the TNFR family, with emphasis on 4-1BB/4-
1BBL, and how these molecules modulate T cell and DC biology, particularly in the context of
viral infection.
1.2 The innate immune response in anti-viral immunity
Viral insult can result in acute infection, e.g. influenza in humans and LCMV Armstrong in
mice, or chronic infection, e.g. human immunodeficiency virus (HIV) in humans, and LCMV
Clone 13 in mice. Moreover, the etiology of one in five (1) of all cancers, as well as several
autoimmune diseases, is also thought to involve viral infection. Both the virulence of the virus
and the magnitude and nature of the immune response against it ultimately determine the
decision between viral clearance and establishment of chronicity. During acute viral infection,
the arms race between the virus and the host results in either elimination of the virus and
generation of immune memory, or death of the host. If the virus cannot be eliminated and does
not kill the host, then both the virus and the immune response must strike a balance between
3
factors that enhance immunity, in order to keep viral replication in check, and factors that slow
down immunity, in order to avoid destruction of self tissues (1, 2). Viral genes that mediate
virulence and cell tropism, as well as multiple immune and non-immune cell types expressing
membrane-bound and soluble molecules act in concert to mediate these outcomes.
Upon viral infection, the innate immune system rapidly induces barriers that control viral
dissemination. Whereas the adaptive immune system generates antigen-specific receptors using
gene rearrangement, the innate immune system uses an array of germ-line encoded receptors
with limited diversity to recognize common features of pathogens (3). Viral components such as
genomic DNA, single-stranded RNA (ssRNA), double-stranded RNA (dsRNA), RNA with 5’-
triphosphate ends and viral proteins are recognized both inside and outside the cell by 3 main
categories of pattern recognition receptors (PRRs): Toll-like receptors (TLRs), retinoic acid-
inducible gene I (RIG-I)-like receptors (RLRs), and nucleotide oligomerization domain (NOD)-
like receptors (NLRs) (4). TLRs are expressed on the cell surface or in endosomal compartments
and signal via adapter proteins such as MyD88, Toll/IL-1R (TIR) domain-containing adapter-
inducing interferon-β (TRIF), TIR domain-containing adapter protein (TIRAP), and TRIF-
related adapter molecule (TRAM), to activate NF-κB and MAPK pathways, and induce the
production of IFNs and other cytokines (5, 6). RLRs are a family of cytoplasmic PRRs that also
drive IFN production and proinflammatory cytokines via signaling pathways involving NF-κB,
IRF3 and IRF7 (7-9). NLRs are a large family of cytosolically expressed PRRs among which are
members that induce the inflammasomes, and regulate maturation of IL-1β and IL-18 via
activation of caspase-1 (7, 10, 11). These receptors are expressed by both immune and non-
immune cells and overall, their signaling leads to secretion of type I IFNs, and other
proinflammatory cytokines and chemokines, as well as increased expression of costimulatory
molecules such as CD80, CD86, and CD40, which are required along with antigen-specific
signals to initiate adaptive immune responses mediated by T and B cells (4).
The cells that make up the innate immune system are highly diverse; they include granulocytes,
such as neutrophils, basophils and eosinophils, lymphocytes such as NK and NKT cells, and
antigen-presenting cells (APCs), such as macrophages and DCs. This thesis focuses on the role
of DCs and NK cells in anti-viral immunity, and these cell types will be discussed further.
1.2.1 Dendritic cells
4
DC development and homeostasis
DCs are specialized APCs that are essential mediators of immunity and tolerance. For his
discovery of DCs, Ralph Steinman received the 2011 Nobel Prize in Medicine and Physiology.
Monocytes can develop some of the features of DCs under conditions of inflammation in vivo or
when cultured with cytokines in vitro, however, they are not precursors for lymphoid organ DCs
in the steady state (12-16). In the bone marrow (BM), monocyte and DC progenitors (MDPs)
diverge to become monocytes and committed DC progenitors (CDPs), which then differentiate
into pre-DCs. Committed pre-DCs leave the BM and migrate to lymphoid and non-lymphoid
tissues via the blood, where they differentiate into the 2 main populations of DCs in the
lymphoid tissues as well as the non-lymphoid tissue CD103+ DCs. This developmental pathway
is highly dependent on Flt3-Flt3L interactions (16). DC homeostasis is maintained by a balance
of 3 factors: continuous input of pre-DCs from the blood, a limited amount of DC division in
situ, and cell death (16). 5% of DCs in lymphoid tissues are dividing at any time (17-19), and in
the steady state this division is regulated by Flt3 (20, 21) and by LTβR signaling, especially on
CD8- spleen DCs (22). DC half-life varies from 5-7 days in the spleen, lymph nodes (LNs), liver
and kidney, and can be as long as 25 days in the lung (17, 19).
DC subsets
DCs are largely divided into conventional DCs (cDCs) and plasmacytoid DCs (pDCs), and both
types can be found in lymphoid tissues, such as spleen and LN, and in non-lymphoid tissues,
such as skin, liver, and lung (16). The cDC subsets in the spleen include the CD8+CD205
+
subset, which is localized primarily in the T cell area and the marginal zone (23), and the CD8-
33D1+ subset, which is found in the red pulp and bridging channels (16). The former is the
primary cell type which captures and cross-presents antigens from apoptotic cells (24-27), while
the latter is more efficient in processing antigens for presentation by MHC-II (28, 29). LNs
contain similar subsets of DCs (CD8+CD205
+ and CD8
-CD11b
+) (24, 30, 31), as well as
migratory DCs that enter the LNs from tissues in a CCR7-dependent manner (32). Antigens can
also enter the LNs directly through the lymph. It is thought that LN-resident DCs that acquire
antigen directly from the lymph are the first to present peptides to naive CD4 T cells, which
results in T cell priming and IL-2 production. Migratory DCs which pick up antigen in the
5
periphery and transport it to LNs then further activate these CD4 T cells to induce differentiation
into effector cells (33).
The differential expression of CD303, CD1C and CD141 divides human DCs into different
subsets (34). Human CD141+ DCs share with mouse CD8
+ DCs the high capacity to mediate
cross-presentation (35, 36) and the CD1C+ subset most closely resembles the mouse CD8
- DC
subset (37, 38). Like mouse pDCs (CD11clo
B220+Ly6C
+PDCA-1
+), human CD303
+ pDCs
secrete large amounts of type I IFNs in response to viral infection (39-42). Studies on human
DCs are hampered by the low frequency of circulating DCs, and therefore most studies have
been conducted using peripheral blood monocytes treated with cytokines to differentiate into
DCs. These in vitro conditions might induce selective DC subsets that exhibit markers and
functions distinct from their physiological counterparts (43).
Functions of DCs
CD8+ DCs are potent producers of IL-12, polarize Th1 responses via production of IL-12, IL-18,
TNF and type I IFNs, and have been shown to be the most important DC subset involved in
cross-presentation. CD8-
DCs produce less IL-12 and polarize Th2 responses when there are
comparatively less of the aforementioned cytokines and in the presence of IL-10. DC-secreted
IL-6 is involved in the differentiation of Th17 cells, and the differentiation of other CD4 T cell
lineages, such as T follicular helper cells (Tfh) and T regulatory cells (Tregs), are also influenced
by DC cytokines (44-46). Both CD8+ and CD8
- DCs are capable of priming efficient CD8 T cell
responses (47-49).
DCs are present in nearly every tissue of the body, especially near portals of entry, where they
survey the environment and act as sentinels of invading pathogens. In addition to initiating
immune responses, DCs are also responsible for inducing central and peripheral tolerance to self-
antigens. In the steady state, most DCs exist in an “immature” or naive state, which is
characterized by low expression of costimulatory molecules, low surface MHC-II, and a high
capacity for phagocytosis and endocytosis. When T cells recognize self-antigen on DCs in this
state, they are either anergized, deleted, or differentiate into Tregs via secretion of cytokines such
as TGFβ (45, 50). The importance of DCs in this process is highlighted by the finding that mice
lacking both cDCs and pDCs develop systemic autoimmunity (51).
6
Following recognition of molecular patterns associated with infection and/or injury/stress, DCs
are activated and undergo a process termed maturation, in which they alter their expression of
chemokine receptors and migrate to lymphoid tissues, upregulate costimulatory molecules and
MHC-II, secrete immunomodulatory cytokines and chemokines, and downregulate their ability
to mediate phagocytosis/endocytosis. CD4 and CD8 T cells that recognize specific antigen on
activated DCs are themselves activated, differentiate, and acquire effector functions (45, 50).
Other cell types such as CD4 T cells can further activate DCs and potentiate immune responses
by upregulating CD40L, which in turn signals through CD40 on DCs. This CD40-mediated
reciprocal activation (DC licensing) has been shown to be important for the ability of DCs to
prime CD8 T cells (52-55). In a recent study (56), it was shown that LTβR signaling in DCs
mediated by lymphotoxin (LT) expressed on activated CD4 T cells also contributes to DC
licensing. LTβR signaling in the DCs induced type I IFN expression, which enhanced the ability
of DCs to stimulate clonal expansion of CD8 T cells. CD40 signaling, on the other hand, was
shown to be more important for the ability of DCs to activate IFNγ production by CD8 T cells
(56, 57). DCs also contribute to humoral immunity via directly presenting unprocessed antigens
to B cells, and by activating helper CD4 T cells (58-61).
Overall, the nature of the signals provided by different microbes, the resulting
cytokine/chemokine milieu, the subset of responding DCs, and the interactions of DCs with other
immune cells ultimately dictates the nature of the immune response.
1.2.2 DCs in anti-viral immunity
As discussed above, DCs enhance immunity to pathogens, including viral pathogens. Due to
their presence at every possible entry site to the body, cDCs are amongst the first cells
encountered by most viruses (62). Among the most studied receptors for viral recognition on
human DCs is DC-SIGN (63, 64). This receptor binds to N-linked glycan substituents on viral
spikes, thereby enabling viral entry (65). It is not clear whether this receptor is actually a pattern
recognition receptor, or whether viruses simply utilize this receptor for entry (62). DEC-205 is
another interesting receptor mediating viral entry because of the enhanced ability of antigens
targeted to this receptor to induce priming of naive T cells (66, 67). Although DEC-205 has been
shown to bind to CpG oligonucleotides (68), and to recognize ligands expressed on dying cells
(69), the precise nature of its natural ligands are not known (69, 70). Other receptors include
7
CD4 and CXCR4 used by HIV, langerin, Fcγ receptors, mannose receptors, and heparan sulfate
(71-73). The fate of viruses after entry can depend on the receptors to which they bind.
In addition to producing large amounts of type I IFNs in response to viral recognition by
endosomal TLRs, pDCs are also capable of cross-presenting viral antigens and rapidly priming
CTLs (74). Most studies in vivo indicate a negative correlation between pDC activity and
severity of viral disease (62, 75). Due to their central role in viral defense, several viruses
downregulate the activity and number of DCs. Viral replication in DCs can directly inhibit their
ability to mature and upregulate key surface markers involved in APC function, and can also
affect the ability of hematopoietic progenitors to develop into DCs (76). Viruses can also affect
the ability of DCs to polarize a Th1 response by downregulating production of cytokines such as
IL-12, thereby favouring a Th2 response (77-79). Th2 responses are known to have detrimental
effects during viral infection by promoting inflammation, allergy, and fibrosis (62). Since DCs
migrate from portals of entry to the body to LNs, some viruses also exploit this mechanism to
disseminate within the host. Lentiviruses especially get concentrated into and/or onto DCs at
mucosal sites, and are not fully digested and presented, but instead shuttled to LNs, where they
infect target cells (80-82).
1.2.3 Therapeutic applications of DCs: DC Vaccination
Owing to their ability to act as natural adjuvants and initiate immune responses, DCs have been
utilized in vaccination strategies aimed at eliciting immune responses against tumors and viruses.
In the clinical setting, peripheral blood monocytes from the patient are differentiated into DCs ex
vivo using a cocktail of cytokines, including GM-CSF and IL-4, loaded with tumor-specific or
virus-specific antigens, matured with DC activation-inducing cytokines or pathogen-associated
molecular patterns (PAMPs), and then injected back into the patient with the hopes of inducing
tumor-specific effector T cells capable of reducing tumor mass, or virus-specific T cells capable
of either protecting a naive individual from serious infection or therapeutically reducing viral
load (62, 83). Clinical studies from the past two decades have explored different DC vaccine
preparations, DC activators, antigen preparations, and routes of DC injection. Thus far, only
Sipuleucel-T, which consists of the patient’s enriched blood APCs cultured with a fusion protein
of prostatic acid phosphatase (an antigen expressed in prostate cancer tissue) and GM-CSF, has
been approved by the US Food and Drug Administration for treatment of metastatic prostate
8
cancer (84). When injected into patients, this drug was shown to extend median survival by
around 4 months (84). There are two ongoing phase III clinical trials evaluating the efficacy of
monocyte-derived ex vivo generated DC vaccines. These trials are testing DC vaccination in
patients with newly diagnosed brain tumors (NCT00045968; Northwest Therapeutics) and in
patients with advanced kidney cancer (NCT01582672; ADAPT trial, Argos Therapeutics) (84).
DC vaccination for the treatment of human viral disease is still being evaluated in animal models
and clinical trials. Studies in animal models of HIV using DCs loaded with HIV-1 viral lysate,
envelope glycoproteins, or inactivated virus have shown that these DCs can mount a potent
immune response against HIV-1 (85-88). In a recent clinical trial, it was shown that autologous
monocyte-derived-DCs pulsed with autologous heat-inactivated whole HIV increased HIV-
specific T cell responses and significantly reduced viral load following combination
antiretroviral therapy interruption (89). What has proven difficult in the field of DC vaccination
is developing efficient protocols that generate large amounts of DCs with the appropriate
maturation status (62).
1.2.4 Natural killer cells
NK cell development and homeostasis
NK cells belong to the family of innate lymphoid cells (ILCs) that are characterized by their
lymphoid morphology, the absence of recombination-activating gene (RAG)-dependent antigen
receptor rearrangement and the absence of myeloid/DC phenotypical markers. Due to their
cytotoxicity and production of IFNγ, NK cells are classified as group 1 ILCs, in contrast to group
2 ILCs, which can produce type 2 cytokines, i.e. IL-5 and IL-13, and group 3 ILCs, which can
produce IL-17 and/or IL-22 (90). NK cells develop and mature in the BM and have been shown
to play a role in the early control of viral infection, tumor immunosurveillance, solid organ and
stem cell transplantation, reproduction, autoimmunity, and asthma (91). The earliest NK cell
progenitors are defined by their potential to respond to IL-15 via upregulation of CD122 and
CD132, and the absence of lineage-specific surface antigens (92, 93). In vitro studies of NK cell
development have also implicated IL-7, stem cell factor, and Flt3L (93), and studies with the
OP9 stromal cell culture system have shown that NK cells can develop in the presence of
specific Notch ligands, such as members of the Delta and jagged families (94-96). The earliest
9
NK cell defining markers are NK1.1 and NKG2D, followed by the Ly49 receptors (see 1.2.4
“NK cell receptors and ligands”) (97). NK cell receptors must undergo an “education” process in
order to acquire functionality. For example, failure to engage inhibitory receptors during
development can result in peripheral NK cells that are hyporesponsive (98-102); however, the
responsiveness of these cells can be continuously modulated when their activating or inhibitory
receptors are ligated (103). Developing immature NK cells do not possess the ability to produce
cytokines and kill other cells, and these functions are acquired late in development following
maturation. Many cytokines, including IL-2, IL-4, IL-7, IL-10, IL-12, IL-15, IL-18, IL-21, type I
IFN, IFNγ and TGFβ have been shown to influence NK cell maturation (104-107). Although NK
cell development has been best characterized in the BM, the presence of phenotypically
immature NK cells in other tissues such as the thymus suggests that these cells can also mature
outside the BM (108). IL-15 especially has an important role during NK cell development and
continues to impact the homeostasis and survival of peripheral NK cells (109). DCs and
macrophages mediate transpresentation of IL-15 to NK cells in vivo (110-112), and IL-15
functions by increasing the expression of pro-survival Bcl-2 family members (113-115), and at
the same time suppressing the transcription factor FOXO3A and the pro-apoptotic molecule BIM
(114). NK cells are widespread throughout most lymphoid and non-lymphoid tissues, with a
prevalence of around 3% of total lymphocytes in the mouse spleen, and from 2-18% of total
lymphocytes in human peripheral blood (116). BrdU and deuterium-enriched glucose labeling
studies have demonstrated that the half-life of NK cells in the spleen is around 7-17 days and
around 12 days in human blood (92, 117-119). Most splenic NK cells in adult mice do not divide
rapidly under normal conditions (117); however, NK cells do proliferate rapidly in lymphopenic
environments, such as following irradiation or transfer into both RAG-1 and γc-deficient mice
(which lack B, T and NK cells) (117, 120, 121).
NK cell subsets
At least four subsets of mouse NK cells have been described based on their expression of CD11b
and CD27. NK cells differentiate from CD11blo
CD27lo
to CD11blo
CD27hi
to CD11bhi
CD27hi
to
the most mature CD11bhi
CD27lo
(122). The double-positive and most mature subsets show a
comparable capacity to kill target cells and produce IFNγ. The CD11blo
CD27hi
NK cells are
predominantly found in the LNs and BM, the CD11bhi
CD27lo
more so in the spleen, liver, lung,
10
and blood, and the double-positive are more homogeneously distributed (123). The chemokine
receptors, chemokines and adhesion molecules regulating the trafficking of NK cells between
these tissues are multifold. In humans, NK cells can be divided into CD56dim
and CD56bright
NK
cell subsets, which differ in their homing properties (124). The lack of CD56 expression on
mouse NK cells, and the differences in CD11b/CD27 expression on mouse and human NK cells
has made it difficult to make direct comparisons, however it is believed that the CD56bright
human NK cell subset most resembles the CD11blo
mouse NK cell subsets (91).
NK cell receptors and ligands
NK cells recognize their targets via an array of cell-surface activating and inhibitory receptors
and their activation is ultimately determined by the balance of signals of the two kinds of
receptors (Table 1.1). Activating receptors can detect stress-induced self ligands and infectious
non-self ligands. These ligands generally show limited expression on healthy cells, and are
upregulated following cellular pathology. NK cell recognition of these ligands underlies
“induced-self” recognition (125). Activating receptors can associate with adapter molecules that
have immunoreceptor tyrosine-based activation motifs (ITAMs), such as FcεRIγ, CD3ζ and
DAP12. Upon ligand binding and tyrosine phosphorylation of the ITAM, tyrosine kinases such
as Syk and ZAP70 are recruited via their SH2 domains and mediate downstream events,
including calcium influx, degranulation and transcription of chemokines and cytokines (126).
Below, I will briefly discuss the activating receptors NKG2D and the natural cytotoxicity
receptors (NCRs).
NKG2D is a type II transmembrane glycoprotein that is expressed as a disulfide-linked
homodimer. There are two isoforms of NKG2D generated by alternative splicing in mice:
NKG2D-L (longer isoform), which recruits the adapter molecule DAP10, and NKG2D-S
(shorter isoform), which recruits either DAP10 or DAP12 (127, 128). NKG2D can be expressed
as both isoforms in mice, and can therefore signal via both DAP10 and DAP12, whereas human
NKG2D is only expressed as the long isoform that signals via DAP10 (129). DAP12 signals via
an ITAM in its cytoplasmic tail; however, DAP10 contains a Tyr-x-x-Met motif (where x
represents any amino acid), which binds either Grb2 or the p85 subunit of phosphatidylinositol-3
11
This table was modified and reproduced from (130)
12
kinase (PI3K) following phosphorylation (131, 132). NKG2D recognizes multiple ligands in
both mice and humans that are homologous to MHC-I. Human NKG2D ligands include MICA,
MICB and six ULPB/RAET1 proteins, and mouse ligands include five isoforms of RAE-1, 3
isoforms of H60, and MULT1 (133).
Normal healthy cells generally show no or very little expression of NKG2D ligands, and these
ligands are only upregulated following infection, transformation, injury, or other forms of stress.
These processes lead to the activation of cellular stress pathways such as the DNA damage
response and the heat shock pathway, but also trigger NF-κB activation, which have all been
shown to regulate the expression of NKG2D ligands (133). There are stringent regulatory
mechanisms at the transcriptional, translational, and post-translational level controlling the
expression of these ligands, ensuring that only unwanted cells are removed. For example,
MULT1 transcripts are abundant in several normal tissues, especially in the thymus (134, 135),
but do not accumulate at the cell surface due to the protein’s ubiquitination and lysosomal
degradation during steady state conditions (136). This process is regulated by 2 closely related
ubiquitin ligases, membrane-associated RING-CH (MARCH) 4 and 9 (137). Downregulation of
MULT1 is prevented in response to stress imparted by heat shock and UV irradiation (136). In
chapter 3, I show that MULT1 is upregulated on B cells following LCMV clone 13 infection.
The activating natural cytotoxicity receptors (NCRs) are type I integral proteins belonging to the
Ig-superfamily. NCRs, including NCR1/NKp46 in mice, and NKp46, NKp30 and NKp44 in
humans, signal via ITAM-containing adapter molecules (138). Several ligands for these
receptors have been identified (see Table 1.1 for receptor/ligand pairs), including viral
hemagglutinin (139), heparan sulfate proteoglycans (140) and nuclear factor BAT3, which can
be released from tumor cells (141). NCRs have been implicated in anti-tumor immunity as NCR
deficiency compromises NK cell cytotoxicity against tumor targets in vivo (142, 143).
Inhibitory receptors that recognize self-MHC molecules (classical and non-classical) or non-
MHC self molecules act to ensure tolerance to self. Viruses have evolved mechanisms to avoid T
cell-mediated killing by downmodulating surface MHC-I expression; however, by so doing they
trigger NK cell activation, as inhibitory receptors on NK cells recognize MHC-I proteins and
other self molecules indicating a healthy cell. In this way, NK cells are thought to recognize
“missing self” on hematopoietic cells (144, 145). Inhibitory receptors signal via immunoreceptor
13
tyrosine-based inhibitory motifs (ITIMs) in their cytoplasmic tails. Phosphorylation of the
tyrosine in the ITIM results in the recruitment of phosphatases, including the tyrosine-specific
phosphatases SHP (SH2-containing protein-tyrosine phosphatase)-1 and SHP2, and the
phospholipid-specific phosphatase SHIP (SH2-containing inositol polyphosphate 5-phosphatase)
(126). These phosphatases decrease the phosphorylation of several intracellular signaling
proteins, including FcεRIγ, ZAP70, Syk, PLCγ, LAT, SLP76, and Vav-1, to downmodulate or
prevent NK cell effector functions (126).
The MHC-I-specific inhibitory receptors include members of the C-type lectin-like Ly49 dimers
in mice, members of the killer cell immunoglobulin-like receptors (KIRs) in humans, and the C-
type lectin-like CD94-NKG2A heterodimers in both species. MHC-I recognition by Ly49
receptors depends on the presence of a peptide in the MHC-I binding groove (146-148), and the
level of Ly49 expression is influenced by the level of MHC-I expression in the mouse (149, 150)
(see Table 1.1 for Ly49/H-2 receptor/ligand interactions). Of interest to the finding of
constitutive interaction between 4-1BB and 4-1BBL on DCs presented in chapter 2 of this thesis,
is that Ly49 receptors can interact with MHC-I ligands in cis on the same membrane, as well as
in trans between cells. Cis interactions can reduce the capacity for trans interactions, thereby
reducing the signaling threshold required for activation (151, 152). There are also activating
Ly49 receptors, e.g. Ly49D and Ly49H, which associate with DAP12 to signal. In contrast to the
other Ly49 receptors, which bind H-2 molecules, the activating Ly49H receptor binds m157, a
viral glycoprotein expressed on MCMV-infected cells (153, 154). Although KIRs differ
structurally from Ly49 receptors, these families of receptors have analogous functions in humans
and mice, respectively, and are examples of convergent evolution (155). KIRs consist of type I
transmembrane proteins with 2 or 3 Ig-like domains, and either a short or long cytoplasmic tail
(156, 157) (see Table 1.1 for KIR/HLA receptor/ligand interactions). Similar to Ly49 receptors,
inhibitory KIRs signal through an ITIM, and activating KIRs associate with DAP12 to mediate
signaling (158). KIRs and Ly49 receptors, as well as their MHC-I ligands, are highly
polymorphic and are expressed in a mono-allelic manner (159, 160) by a stochastic mechanism
of gene regulation (161), resulting in distinctive subsets of NK cells that have unique properties
and activation thresholds. The CD94-NKG2 heterodimers bind to the non-classical MHC-I
proteins, HLA-E in humans and Qa1b in mice. These ligands could enable the heterodimers to
indirectly monitor the expression of classical MHC-I on target cells as HLA-E and Qa1b bind
14
peptides derived from the leader sequence of classical MHC-I proteins (162, 163).
CD94/NKG2A heterodimers are inhibitory due to the ITIM contained in the cytoplasmic tail of
NKG2A, whereas CD94/NKG2C/E heterodimers can associate with DAP12 and are thought to
mediate activation (158). In contrast to the Ly49 receptors and KIRs, the CD94/NKG2 receptors
have limited polymorphism and are not stably expressed, as their expression can be modulated
by several factors, including the cytokine milieu (126, 164).
Non-MHC-I inhibitory receptors include the NKR-P1 receptors, which recognize Clr ligands in
mice, NKR-P1A, which recognizes LLT1 in humans, and 2B4, which recognizes CD48 in both
species. The C-type lectin-like NKR-P1 receptors can be either activating or inhibitory and bind
to a family of genetically linked C-type lectin-like ligands, Clr proteins (165). LLT1 is a
functional homologue of mouse Clr in humans (166, 167). The receptor/ligand interactions and
subsequent signaling within the NKR-P1/Clr system were recently reviewed in (165), and the
inhibitory NKR-P1B receptor is briefly discussed in section 1.2.5 of this thesis. 2B4, and its
ligand CD48, are members of the CD2 family of Ig-related proteins. The cytoplasmic tail of 2B4
contains an immunoreceptor tyrosine-based switch motif, which can recruit tyrosine
phosphatases, i.e. SHP-1 and SHP-2, and the adapter proteins SAP, EAT2 or ERT, upon tyrosine
phosphorylation (126, 168, 169). Signals mediated by 2B4 can be either activating or inhibitory,
depending on the isoform of the receptor, the recruited adapter proteins, the species, as well as
the stage of NK cell differentiation and activation (126, 158).
Functions of NK cells
NK cell activation is itself modulated by the cytokine microenvironment and also contributes to
the modulation of other cell types. Cytokines such as Type I IFN, IL-2, IL-12, IL-18 and IL-15
produced by other cell types are potent activators of NK cell effector function (91). Upon
activation, NK cells kill target cells via exocytosis of cytoplasmic granules containing perforin
and granzymes, FasL-mediated induction of apoptosis, antibody-dependent cellular cytotoxicity
(ADCC), and secrete cytokines such as IFNγ and TNF (91). NK cells also interact with other
cells of the immune system. IFNγ and TNF secreted by NK cells can promote the maturation of
DCs, which in turn can further activate NK cells via DC-derived IL-12 or IL-18 (170-172).
However, NK cells have also been shown to kill DCs, thereby influencing DC homeostasis
(173). In addition to NK cell-derived IFNγ promoting the priming of Th1 cells in the inflamed
15
LNs (174, 175), NK cells have also been shown to kill activated T cells under certain conditions,
which will be discussed in section 1.5.4. In addition to direct lysis, NK cell acquisition of MHC-
II molecules from DCs has been shown to reduce CD4 T cell responses (176). In the Fas-
deficient mouse model, NK cells were shown to suppress autoreactive B cells in vitro, and NK
cell depletion in vivo worsened the severity of autoimmunity (177). Overstimulated macrophages
have also been shown to be targets of NK cell cytotoxicity (178). In sum, there is evidence that
in addition to their well known role as part of the innate activation of the immune system, NK
cells are also important in immune system downregulation, by eliminating activated cells.
The above mentioned innate effector functions of NK cells are well appreciated; however, recent
evidence in mice and humans suggests that NK cells also exhibit adaptive features, such as
antigen-specificity, clonal expansion, and the ability to generate long-lived memory cells that re-
expand and protect the host following re-exposure. Although the precise phenotype of these
memory NK cells remains elusive, they have been defined as showing relatively increased
expression of Ly49H, Ly6C, CD43, and KLRG1, and decreased expression of CD27 in the
MCMV system (179).
1.2.5 NK cells in anti-viral immunity
NK cells contain viral replication by killing infected cells, thereby controlling viral spread early
following infection, prior to the effector functions of adaptive immunity. A protective effect of
NK cells has been described in HSV-1, influenza virus, ectromelia poxvirus, vaccinia virus
(VV), HIV, hepatitis C virus (HCV), and hepatitis B virus (HBV) infections, and especially in
murine cytomegalovirus (MCMV) infection (180, 181). NK cell depletion is associated with
increased susceptibility to MCMV, and NK cell adoptive transfer with resistance to MCMV
infection (182, 183). Influenza infection of NK cell-depleted mice or of mice with defects in NK
cell activity results in delayed clearance of virus from lungs, and enhanced morbidity and
mortality (184, 185). Simian immunodeficiency virus (SIV) infection of rhesus macaques and
population genetic studies of HIV infection in humans have also suggested a role for NK cells in
modulating the outcome of HIV disease (186-190). Further evidence supporting a protective role
of NK cells comes from the multiple examples of viruses elaborating mechanisms to evade the
NK cell response. For example, MCMV encodes genes (e.g. MCMV m145, m152, m155) that
downregulate the expression of NKG2D ligands, thereby preventing NK cell activation, and also
16
encodes genes (e.g. MCMV m144) that act as MHC-I homologs, thereby binding/signaling
inhibitory receptors on NK cells (191). Upon infection with rat CMV (RCMV), infected cells
downregulate Clr-b, which is a C-type lectin-like protein that binds to the inhibitory NK cell
receptor NKR-P1B. Thus, Clr-b downregulation sensitizes infected cells to killing by NK cells.
However, RCMV encodes a decoy gene product (RCTL) that has homology to the Clr ligands
and when expressed, protects infected cells from NK cell killing via interaction with NKR-P1B
(192). Cell-surface expression of Clr-b has also been shown to be downregulated in murine cell
lines and BM-derived macrophages by infection with two poxviruses, ectromelia virus and VV
(193). The precise mechanisms underlying Clr-b downregulation following viral infection are not
known but could involve the activation of cellular stress pathways, which have been shown to
cause downregulation of Clr-b at the cell surface (194). It is also possible that the virus encodes
proteins that affect Clr-b protein trafficking and/or turnover.
NK cells have also been shown to perform regulatory activities during viral infection, thereby
dampening anti-viral immunity, and promoting viral persistence (180). These functions of NK
cells might have evolved to control virus-mediated immunopathology and will be discussed in
the section on the role of NK cells during LCMV infection (1.5.4).
In sum, the activation of innate immune cells, such as DCs and NK cells, and their subsequent
effector functions result in early viral recognition, killing of infected cells, and initiation of
antigen-specific adaptive immune responses. However, it is becoming increasingly appreciated
that these innate cells can also serve a regulatory function and can continue to be key players
throughout the course of viral infection (195, 196).
1.3 The spleen as a secondary lymphoid organ involved in anti-viral immunity
Immune responses against pathogens, such as viruses, are orchestrated in secondary lymphoid
organs. The spleen is the largest secondary lymphoid organ and is situated in the left cranial
abdomen, below the diaphragm and connected to the stomach. Its main functions are to remove
aging erythrocytes, filter soluble and particulate antigens from blood, and initiate immune
responses to blood-borne microorganisms (197).
1.3.1 Development of the spleen
17
The formation of the splanchnic mesodermal plate (SMP) at E12 is the initial event in the
development of the spleen. The SMP acts as an anlage to organize further formation of the
spleen (198). When formation of the SMP is defective, as occurs in mice that have a mutation in
the dominant hemimelia gene (199), or the cells that form it fail to proliferate, spleen
development halts. Several transcription factors such as homeobox 11 (Hox11), Wilm’s tumor 1,
Pbx1, Nkx3.2 and Pod1 are absolutely necessary for spleen formation (200-204). Genetic
experiments have shown that Pbx1 interacts with Hox11, and appears to be required earlier than
Nkx3.2 and Pod1 (204).
Progenitors of the erythroid and myeloid lineages are the first to colonize the spleen. On day
E13.5, lymphoid tissue inducer cells (LTi) are detectable in the spleen (205), and the first
hematopoietic stem cells (HSCs) appear at day E14.5 (206). The process of lymphoid
organogenesis has perhaps been best studied in LNs. During LN and Peyer’s patch (PP)
development, stromal lymphoid tissue organizer cells (LTo) expressing LTβR engage with
LTα1β2 expressed by LTi to activate classical and alternative NF-κB, resulting in the expression
of VCAM-1 and ICAM-1 on LTo and the production of CCL19, CCL21, and CXCL13. These
signals, along with TNFR1 signaling, increase attraction and retention of LTi and other
hematopoietic cells in the developing LN anlagen and induce further LTα1β2 expression, creating
a positive feedback loop that sustains LN development (207, 208).
While the development of LNs has been fairly well studied, the picture is less clear in the spleen.
Although LTi are present in neonatal spleen, they do not express LTα1β2, and the earliest
formation of white pulp and compartmentalization of red and white pulp in the spleen is LTα1β2-
independent. However, postnatal development of the white pulp, which involves the influx of T
cells, depends on LTα1β2 expression by B cells, which occurs starting around 4 days after birth
(209). At midgestation, spleen mesenchymal cells include LTo (209-212), but whether these
cells function like their LN counterparts in organizing the formation of lymphoid compartments
is not well known (213).
Overall, there are many gaps in the knowledge of mechanisms governing the development and
organization of the spleen, but it is clear that the molecular interactions governing these
processes are distinct from those involved in the generation of LNs and PPs (198). For example,
aly/aly mice, which have a point mutation in the alternative NF-κB-inducing kinase (NIK) (214),
18
or mice deficient in LTα, lack all LNs and PPs, but still have a spleen, albeit with a disturbed
structure (215, 216).
1.3.2 Structure and function of the spleen
The spleen is a highly organized organ that is separated into areas of red pulp and white pulp
(Fig. 1.1). The red pulp largely consists of macrophages that are involved in iron metabolism and
removing old RBC and bacteria from the blood, and plasmablasts/plasma cells producing
antibodies. The white pulp, or the lymphoid region, of the spleen consists of distinct T and B cell
areas, surrounded by the marginal zone. Hematopoietic cells from the blood enter the white pulp
via the marginal zone, which consists of SIGNR1/MARCO-expressing marginal zone
macrophages, SIGLEC1-expressing marginal zone metallophilic macrophages, marginal zone B
cells, and DCs. The main difference in the structure of the mouse and human spleen is that
humans have an inner and outer marginal zone, which is surrounded by a large perifollicular
zone containing macrophages (198, 217).
The organization of the white pulp is maintained by specific chemokine gradients and cell-cell
interactions. B cells express the chemokine receptor CXCR5, which mediates their migration to
CXCL13, produced by follicular DCs (FDCs) and other stromal cells in the B cell follicles (207).
T cells and DCs express the chemokine receptor CCR7, which mediates their migration to the
ligands CCL19 and CCL21, produced by stromal cells in the T cell zone (218). The expression
of these chemokines is regulated by LTβR and TNFR1 signaling between stromal cells and
hematopoietic cells; disorganization of the white pulp occurs in the absence of these receptors
(219). These molecules are involved in the activation of transcription factors in the NF-κB
family. For example, triggering LTβR results in the expression of CCL21 in the spleen via
activation of the alternative IKKα/NIK-dependent NF-κB pathway (208).
Owing to their expression of an array of pattern recognition receptors and scavenger receptors,
marginal zone macrophages are potently involved in the uptake of bacteria and viruses in the
bloodstream, and produce cytokines such as type I IFNs. Marginal zone B cells are specialized to
detect blood borne pathogens and quickly differentiate into IgM-producing plasma cells, and
along with migratory and splenic DCs, can migrate to the white pulp to function as APCs.
Activated T cells and B cells meet at the T-B border to initiate adaptive immune responses. The
19
This figure was adapted from (217, 220)
20
precise mechanisms governing lymphocyte exit from the white pulp are unclear but likely
involve the modulation of cell surface chemokine receptors (198, 217).
The central function of the spleen in protection from encapsulated bacteria was highlighted in
studies of splenectomized mice and humans. Although the spleen is involved in anti-viral
immunity, it seems to be less vital than other secondary lymphoid tissues, such as LNs, in
mediating clearance of certain viruses. LCMV Armstrong or LCMV WE infection of mice
lacking a spleen, Hox11-/-
mice, resulted in barely detectable levels of virus and viral clearance
by day 15 at the latest. However, LCMV infection of aly/aly mutant mice, which lack all
secondary lymphoid organs (e.g. LNs and PPs), except the spleen, resulted in life-long viral
persistence. Similarly, infection of aly/aly mice with VV and vesicular stomatitis virus (VSV)
resulted in significantly delayed viral clearance and no production of neutralizing IgG,
respectively, whereas these infections in Hox11-/-
mice resulted in no abnormalities following
VV infection, and only slightly delayed antibody production following VSV infection. Aly/aly
mice succumbed to lethal encephalitis following VSV infection due to the lack of a neutralizing
IgG response, while Hox11-/-
and WT mice were protected from encephalitis. It should be noted
that although aly/aly mice contain a spleen, it exhibits a disturbed structure, which certainly
complicates the interpretation of studies with these mice (221). In contrast to these studies
however, infection of mice lacking splenic macrophages with acute LCMV and VSV was shown
to impair viral control, suggesting that the spleen or at least cell types in the spleen are important
in the defense against these viral pathogens (222, 223). It is possible that other factors in Hox11-/-
mice or differences in strains and doses of virus might account for these differences. Thus, the
indispensable role of the spleen in certain bacterial infections is clear, however its absolute
requirement during anti-viral immunity is less so.
1.4 Splenic atrophy due to viral infection and other causes
Splenic atrophy refers to the wasting of the spleen, and can result in hyposplenism, reduced
splenic function, or asplenia, a non-functional spleen. Splenic atrophy can result in compromised
immunity and compromised blood filtering. In humans, splenic atrophy and
hyposplenism/asplenia are commonly assessed by CT scan of the abdomen, a technetium-99
scan, and the presence of Howell-Jolly bodies, which are nuclear remnants in RBC normally
removed by the spleen. Acquired splenic atrophy in humans is most commonly associated with
21
sickle cell anemia (224, 225), but also with autoimmune conditions (226, 227), such as celiac
disease (228-231), systemic lupus erythematosus (SLE) (227, 232) and Sjögren’s syndrome (227,
233). Although viral infections can induce transient splenomegaly due to an active immune
response in the spleen, infection with severe acute respiratory syndrome (SARS) (234) and
H5N1 (235) in humans, as well as coxsackievirus B3 (236), certain strains of parainfluenza virus
(237), murine gammaherpesvirus 68 (MHV68) (238, 239), and tick-borne encephalitis virus
(TBEV) (240) infections in mice have been associated with atrophy of spleen tissue.
1.4.1 Splenic atrophy in sickle cell anemia and autoimmunity
The pathophysiological mechanisms underlying splenic atrophy are not understood in most
cases. In sickle cell anemia, it is thought to occur due to repeated attacks of vasooclusion and
infarction within the splenic microvasculature, eventually leading to autosplenectomy in some
individuals (224). Splenic atrophy in intestinal disorders such as celiac disease has been
associated with folate deficiency (226), but also with autoimmune reactions leading to loss of
cells in the spleen. Celiac disease is frequently associated with a number of other autoimmune
diseases such as Sjögren’s syndrome and SLE, and it is not entirely clear whether splenic atrophy
predisposes to autoimmunity, or vice versa (228, 241, 242). Marginal zone B cells and DCs play
a role in the tolerance of autoantigens, and favour the expansion of Tregs (243-245). If these
cells are reduced in number or have compromised function as can occur following splenic
atrophy/hyposplenia, it is conceivable that this can promote the development of autoimmunity
(229). In SLE, splenic atrophy and hyposplenism have been linked to immune complex
saturation, leading to Fc-receptor blockage, as well as microthrombosis in the spleen related to
anti-phospholipid antibodies (227, 232).
1.4.2 Splenic atrophy due to viral infection: what are the mechanisms?
Splenic atrophy following viral infection is also poorly understood and has been attributed to the
cytopathicity of the virus itself, the resulting production of proinflammatory cytokines such as
TNF, the effector functions of T cells, and activation of the sympathetic nervous system.
Atrophy of the white pulp of the spleen following SARS infection in humans and parainfluenza
strain YN infection of mice is thought to be due to direct injury of the splenocytes by the virus
(234, 237). In the SARS study, they also found that the extent of damage to the immune cells
22
was related to the length of the disease, in that a longer duration conferred more severe damage.
In contrast, splenic atrophy induced by infection of mice with coxsackievirus B3 and TBEV was
shown to occur in the absence of detectable virus in the cells being depleted (236, 240). Splenic
atrophy in the absence of direct viral cytopathicity of immune cells might be mediated by viral
infection of cell types required for lymphoid tissue homeostasis or viral factors that directly
interfere with immune cell production/maturation, diverted homing of lymphocytes to
nonlymphoid organs, autoreactive phenomena triggered by infection, or the depletion of immune
cells by the immune response generated by the virus.
In the murine models of splenic atrophy discussed above, the spleen was not the only organ
affected, as an involuted thymus and smaller LNs were also evident in some models (236, 237,
240). Loss of spleen size was also accompanied by a reduction in body weight, although this was
considered not to explain atrophy of the spleen, as loss in bodyweight was disproportional with
the loss in organ weight (236). Atrophic spleens were shown to be approximately 2-3 fold
smaller than uninfected controls in size (236, 237) and number of immune cells (236, 240), and
the kinetics of atrophy was quite fast following infection, with an onset of atrophy between 5-6
days following coxsackievirus B3 infection (236). Splenic atrophy following parainfluenza strain
YN and TBEV infection was assessed at only one time point, 7 and 13 days post-infection (dpi),
respectively (237, 240). In those studies where the number of specific cell types in the spleen
was assessed, it was shown that T cell, B cell and null subsets were affected (236, 239, 240).
Atrophy was irreversible as these infections ultimately killed their hosts (236, 237, 240). This,
however, was not the case with MHV68 infection of IFNγR-/-
mice (239).
MHV68 infection of WT and IFNγR-/-
mice results in similar viral clearance from the lung, and
both groups of mice do not succumb to the infection. However, unlike infection of WT mice,
infection of IFNγR-/-
mice leads to extensive and sustained splenic atrophy, as well as destruction
of splenic architecture. These pathological changes in the spleen were attributed to CD8 T cells,
in that CD8 T cell depletion prevented these changes. How the absence of IFNγ causes the CD8
T cells to induce splenic atrophy is not clear, but is thought to involve the production of perforin
and other factors normally controlled or inhibited in the presence of IFNγR. Virus replication in
splenocytes followed by a lysis of infected cells was eliminated as the mechanism underlying
splenic atrophy in this model, as blocking viral replication at 6 dpi did not impact splenic atrophy
23
that became evident at 14 dpi. This study was taken out to 70 dpi, a time point at which IFNγR-/-
mice still had spleens that were around 2-3 times smaller than WT (238, 239).
Stress responses following viral infection and stroke, associated with increased activation of the
sympathetic nervous system and increased levels of glucocorticoid hormones, are also associated
with splenic/thymic atrophy, as these steroids are anti-inflammatory and can trigger apoptosis in
immune cells through activation of caspases (240, 246, 247).
These studies of splenic atrophy suggest that spleen size is largely determined by the number of
cells within the spleen, and that it is affected by the cytopathicity of the virus, the effector
functions of immune cells such as T cells, cytokine production, the integrity of the vasculature
supporting the spleen, and the stress response. The exact mechanisms underlying splenic atrophy
or its causes are not clear in any of these studies, leaving investigators only to speculate. In
chapter 3 of this thesis, I investigate the mechanisms underlying splenic atrophy following
persistent LCMV clone 13 infection, which will be discussed in section 1.5.
1.4.3 Compromised immunity due to complications associated with the spleen
Pathology of the spleen due to splenic atrophy, hyposplenism, or asplenia is associated with an
increased risk of infection to encapsulated bacteria such as Streptococcus pneumonia, Neisseria
meningitidis, and Haemophylus influenzae. This is due to defective/reduced activity of
opsonizing molecules such as properdin and tuftsin, and because of reduced numbers of natural
antibody producing-IgM-memory B cells in the marginal zone of the spleen, which are
responsible for mediating T-independent responses against these bacteria. Treatments include
prophylactic antibiotic therapy and immunization against these pathogens (248). Increased
susceptibility to the gram-negative bacteria Salmonella has also been documented (249, 250).
It is not entirely clear how much spleen tissue is required to protect against infection (251). Some
studies have investigated this; however, it seems that the precise mass/dimensions probably
depend on the nature of the infection and the immune response against it. In chapter 3 of this
thesis, I investigate the ability of LCMV-infected mice experiencing splenic atrophy to clear
Salmonella infection.
1.5 Murine models of viral infection: LCMV
24
1.5.1 LCMV virus
LCMV is a non-cytopathic rodent pathogen that was first isolated by Charles Armstrong in 1934,
and is the prototypical member of the arenaviridae family (252). It is a spherical, enveloped virus
that consists of two ssRNA segments, designated L (7.2 kb) and S (3.4 kb). The L segment
encodes the viral RNA-dependent RNA polymerase and a RING finger protein called Z, which is
thought to act as a matrix protein (253) and regulate transcription (254). The S segment encodes
the two main structural proteins: nucleoprotein (NP) and the glycoprotein (GP) precursor (GP-
C). GP-C is post-translationally cleaved to GP-1 and GP-2. GP-1 is thought to mediate binding
to the cellular receptor for LCMV, α-dystroglycan (αDG) (255-257). The outcome of LCMV
infection in mice depends on the size of the viral inoculum, the strain of the virus, age of the host
and route of infection.
1.5.2 Armstrong vs. clone 13: mechanisms underlying clearance vs. persistence
There are several strains of LCMV that differ in viral pathogenesis and persistence; of interest to
the present work are LCMV Armstrong (ARM) and LCMV clone 13 (Cl13). The acutely
infecting strain, ARM, is the parental strain, while the persistent genetic variant Cl13 was
isolated from the spleens of 2-month old BALB/c WEHI mice that had been infected with ARM
at birth (258). These carrier mice can make antibody responses to the virus, but diminished CTL
responses, due to the thymic deletion of virus-specific CTL during development (259-263). The
absence of a functional anti-LCMV CTL response in these neonatally ARM-infected mice leads
to life-long infection.
Although ARM and Cl13 differ dramatically in their persistence in the host, in that ARM is
cleared within 1 week, whereas Cl13 can persist for months, they are surprisingly genetically
very similar (264-266). Armstrong and Cl13 differ by only 5 nucleotides, of which 3 result in
amino acid changes. The lysine to glutamine change at position 1079 in the viral polymerase was
shown to selectively increase viral titers in macrophages, but the overall contribution of this
change to viral persistence remains ill-defined (265). The recently identified asparagine (ARM)
to aspartic acid (Cl13) amino acid substitution at position 176 in GP1, present in roughly 50% of
persistent LCMV strains, was shown not to be required for the ability of Cl13 to persist long-
term (267). In contrast, the phenylalanine (F) to leucine (L) substitution at position 260 in GP1,
25
present in more than 95% of viral isolates mediating persistence, was shown to be essential for
the ability of Cl13 to persist long-term (268, 269). Cl13 binds to αDG, the cellular receptor for
LCMV, with 10-1000x higher affinity than ARM, a difference that is associated with the F260L
amino acid substitution, and has profound functional consequences. Among cells of the immune
system, αDG is preferentially expressed on CD11c+/DEC205
+ splenic DCs, and shows very little
expression on T, B, or CD11b+ cells. Strains and variants of LCMV that bind with high affinity
to αDG are associated with replication in the marginal zone and white pulp of the spleen and
viral persistence, whereas those that bind with lower affinity to αDG are associated with the red
pulp and viral clearance. The high expression of αDG on DCs leads to the higher infectivity and
impairment of these cells by Cl13, resulting in long-term persistence (270-272). Cl13 infection
inhibits DC maturation and upregulation of key surface markers involved in APC function
(MHC-I/II, CD40, CD80, CD86), and upregulates immunosuppressive cytokines and ligands,
thereby reducing the ability of these cells to fully prime T cells (76, 273-276). In contrast, ARM
predominantly infects F4/80+ macrophages and a few DCs (270).
There is evidence that LCMV uses receptors other than αDG to infect cells. αDG-/-
mouse
embryonic stem (ES) cells can be infected by non-immunosuppressive strains such as ARM,
which infects more than 50% of these cells by 48 hours following infection (277).
Immunosuppressive variants, such as Cl13, show little infection of αDG-/-
ES cells after 48 hours
(<10%), indicating a strong dependence of infection on αDG. However, the immunosuppressive
variant PBL364 can infect more than 80% of ES cells lacking αDG by 48 hours following
infection (277). Two members of the TAM (Tyro3 Axl Mer) family of receptors (Tyro3 and
Axl), as well as DC-SIGN and lymph node sinusoidal endothelial calcium-dependent lectin
(LSECtin) were recently suggested to act as receptors for LCMV and a related arenavirus, Lassa
virus (278, 279). Jurkat T cells overexpressing Axl, Tyro3, DC-SIGN, LSECtin, or a control
protein were infected with viruses pseudotyped with the GPs of 4 strains of LCMV, including
ARM and Cl13. It was found that in the presence of these receptors on Jurkat cells, there was 2-3
logs higher infectivity when compared to control. There weren’t any striking differences in the
infectivity of immunosuppressive and non-immunosuppressive strains, indicating that usage of
these receptors does not correlate directly with LCMV virulence (278). Following this report,
Sullivan et al. evaluated the physiological relevance of Axl during in vivo ARM and Cl13
infection. Axl-/-
and WT mice cleared Cl13 from the blood with similar kinetics, and there were
26
no quantitative or qualitative differences in the CD4 or CD8 T cell responses following both
ARM and Cl13 infection (280). These findings indicate a lack of major in vivo significance of
Axl expression in the establishment or course of an acute or persistent LCMV infection, although
it is possible that the expression of the other TAM receptors, Tyro3 and Mer, compensate for the
lack of Axl. In sum, αDG is still the only receptor identified that correlates with LCMV
pathogenicity, but it is likely that other receptors exist. The in vivo relevance of Tyro3, DC-
SIGN, and LSECtin during LCMV infection remains to be evaluated.
Following ARM infection, a robust anti-LCMV CTL response is generated that is associated
with specific lysis of infected cells, and production of effector molecules, including IFNγ, TNF,
IL-2, perforin, and granzyme B (281-283). CD8 T cells reactive to the immunodominant LCMV
peptides NP396-404, GP33-41, and to a lesser extent GP276-286 in the context of MHC-I H-2Db
(284-
288), can reach a peak expansion of around 107 cells per spleen by day 8 following infection and
can constitute up to 50% of CD8 T cells in the spleen (266, 289, 290). Following expansion and
viral clearance, more than 90-95% of these cells die (290, 291), leaving behind a small
population of memory CD8 T cells (292, 293). In contrast to ARM infection, infection with Cl13
is associated with physical deletion of NP396-404-reactive CD8 T cells (266, 294, 295) and
functional impairment of other viral-specific CD8 T cells, including those reactive to GP33-41 and
GP276-286. Since NP is the most abundantly expressed viral antigen, those CD8 T cells reactive to
NP396-404 are the ones most likely to be stimulated and eventually deleted/exhausted, whereas
those reactive to the less abundant GP are present and functional longer (296, 297). Loss of
function is indirectly correlated with antigen persistence/viral load. This functional impairment
(exhaustion) is associated with a hierarchical loss of function, with the ability to lyse cells and
produce IL-2 lost first, followed by the inability to produce TNF, and lastly IFNγ (266).
In addition to the loss of effector function, T cell exhaustion following persistent LCMV
infection is also associated with the expression of inhibitory cell surface molecules, including
PD-1/PD-L1, LAG-3, Tim-3, 2B4, CD160, and CTLA-4 and immunoregulatory cytokines,
including IL-10 and TGFβ (298). When comparing functional memory CD8 T cells with
functionally impaired viral-specific CD8 T cells by gene array, it was found that the exhausted T
cells showed enhanced expression of the inhibitory molecule PD-1. Blocking PD-1-PD-L1
interactions in vivo was shown to restore the ability of CD8 T cells in chronically infected mice
27
to proliferate, produce cytokines, kill cells, and decrease viral load (299). Similarly, production
of the immunoregulatory cytokine IL-10 was found to be sustained following persistent LCMV
infection, and IL-10R blockade mediated clearance of an otherwise persistent Cl13 infection
(300, 301). CD4 and CD8 T cells, as well as DCs, macrophages, and NK cells have been
implicated in producing IL-10 during persistent viral infection (302). The above findings have
been corroborated in studies of HIV and HCV infection in humans, suggesting that the
immunoregulatory mechanisms underlying chronic viral infection are shared by distinct viruses
in different hosts. These mechanisms are thought to have evolved to put a break on the T cell
response when virus cannot be cleared, so as to avoid immune-mediated pathology. For example,
PD-L1-/-
mice succumb to Cl13 infection within 8 days due to immunopathological damage
(299).
CD8 T cells are the main cell type involved in viral clearance, as mice lacking CD8 T cells never
purge the virus (303). Several lines of evidence make it evident that CD4 T cells also play a
substantial role. Following ARM infection, virus-specific CD4 T cells, which are primarily Th1
in nature, expand with slower kinetics and to lower numbers than CD8 T cells, and produce IFNγ
and IL-2 (304-307). In the absence of CD4 T cells, mice are capable of clearing ARM infection,
but show evidence of T cell exhaustion and defects in the generation of a stable memory pool
(297, 308, 309). Compared to ARM infection, Cl13 infection induces viral-specific CD4 T cells
that are reduced in number, and show functional defects, such as in production of IFNγ, IL-2 and
TNF (310-312). In this way, similar to CD8 T cells, CD4 T cells also show evidence of
exhaustion during persistent LCMV infection. Nevertheless, these CD4 T cells are critical for
eventual control of the virus, as CD4 T cell depletion during persistent LCMV infection leads to
uncontrolled life-long viremia (313). CD4 T cell help, associated with the production of IL-21,
provided to CD8 T cells and antibody-producing B cells, eventually controls persistent LCMV
infection within 2-3 months in the periphery (314, 315). Signaling through costimulatory
molecules such as OX40 on CD4 T cells has been shown to contribute to control of LCMV clone
13 infection (316).
1.5.3 The innate immune response to LCMV infection
Although much is understood about the role of the adaptive immune response to LCMV
infection, the contribution of innate cell types is relatively poorly defined. DCs are the critical
28
APCs required to mount an effective CTL response following LCMV infection, in that mice
lacking DCs, but not macrophages and B cells, show defective priming (317). Within 24 hours of
ARM infection, splenic DCs produce cytokines including type I IFN, TNF, and IL-12, and
acquire the ability to stimulate naive LCMV-specific CD8 T cells ex vivo (318, 319). This DC
activation is accompanied by an increased tendency to undergo apoptosis, and cDC numbers in
the spleen decrease around 2-fold by 3 dpi. Both DC activation and apoptosis are IFN-dependent.
The decline in cDCs is accompanied by an increase in the number of pDCs by 3 dpi, which also
upregulate type I IFN production by 24 hours post-infection (pi) (318).
In a recent study, the innate immune response to ARM and Cl13 infection was compared side by
side. This study identified no difference in the early response (within 72 hours) of DCs to both
infections, in that DCs similarly upregulated activation markers and stimulated anti-viral CD8 T
cells, although by day 14 pi, myeloid cells from Cl13-infected mice showed an inability to
effectively stimulate T cell responses. By 7 dpi, there was an increase in a population of
myeloid-derived suppressor cells (MDSCs) during both infections in lymphoid organs and blood,
which presumably arise to dampen immune responses and avoid immunopathology. This
population of MDSCs was greater in the Cl13-infected animals, persisted compared to ARM-
infected animals, and potently suppressed T cell proliferation ex vivo. Depleting these cells with
a Gr-1 antibody enhanced anti-viral CD8 T cell responses, but did not affect viral load (195).
Other studies have also identified the enhanced and sustained presence of immunoregulatory
APCs expressing molecules such as IL-10 and PD-L1 during Cl13 infection (275, 320). These
studies highlight the involvement of the innate immune system both early and throughout the
course of persistent viral infection.
The aforementioned differences in the nature of ARM and Cl13 and the immune responses they
elicit make these viruses excellent models to investigate the processes underlying acute versus
persistent viral infection. Studies of LCMV have advanced our understanding of the pathological
mechanisms underlying chronic viral infections and many of the findings have held true in
human infections, such as HIV, HCV, and HBV.
1.5.4 The role of NK cells during LCMV infection
29
In general, LCMV infection is relatively NK-resistant, in that NK cells do not directly interfere
with LCMV replication in the mouse (180). Although IFNγ expressed by NK cells has been
implicated in controlling LCMV replication following ARM infection (321), the data
overwhelmingly point to a role for NK cells not in direct control of virus, but in modulation of
anti-LCMV T cell responses, which then impact viral control. By infecting NK cell-deficient
mice and WT NK cell-depleted mice with LCMV WE, an acute strain of LCMV that is more
persistent than ARM, Lang et al. showed that NK cells mediate viral persistence of LCMV by
killing activated LCMV-specific CD8 T cells in a perforin-dependent manner. In the absence of
NK cells, mice were able to clear the virus in several organs (322).
Waggoner et al. took a more detailed approach and compared low, medium, and high dose
infection with Cl13, and concluded that NK cells act as “rheostats” in controlling anti-viral T cell
immunity. Low-dose (5 × 104
PFU) infection generates a robust anti-LCMV T cell response
which results in viral clearance. In this model, NK cell depletion enhanced the CD8 T cell
response, which had no effect on the already effective clearance of virus. Medium dose Cl13 (2 ×
105 PFU) normally results in partial functional exhaustion of T cells and T-cell dependent
immunopathology. In this setting, NK cell depletion reversed the immunopathology because a
greater number of anti-LCMV CD4 and CD8 T cells were able to enhance viral clearance before
the acquisition of severe immune pathology. However, depletion of NK cells prior to a high dose
infection (2 × 106 PFU), which normally results in a persistent infection not associated with
mortality, worsened the outcome, mediating increased immunopathology and mortality. In this
context, NK cells were required to dampen immunopathological T cell responses. Overall, NK
cell downmodulation of anti-viral T cell responses in the context of LCMV infection can be
either beneficial or detrimental, depending on whether the T cell response is mediating viral
clearance or immunopathology. In contrast to Lang’s study, Waggoner et al. point to a direct
perforin-mediated cytolytic effect of NK cells on CD4 T cells, which then negatively impact the
anti-viral CD8 T cell response (323).
These studies looked at the role of NK cells early following infection, in that mice were depleted
of NK cells prior to infection. A recent study evaluated the outcome of NK cell depletion 1 day-4
weeks following Cl13 infection, and found that NK cells continue to promote persistent viral
infection due to downmodulation of virus-specific T cell responses. The effect of NK cell
30
depletion on viral burden and virus-specific T cell responses was found to be dependent on the
size of the viral inoculum, viral load at the time of depletion, and the presence of CD4 T cells
(196). In contrast to these findings, another report suggested that delayed NK cell depletion
following Cl13 infection did not significantly impact the numbers of virus-specific T cells as
measured by cytokine production (324). It is argued that whether or not late-stage NK cell
depletion impacts anti-viral T cell responses and viral load depends on the viral load and the
extent of immune exhaustion at the time of NK cell depletion. If viral load is high, then anti-viral
T cells are more exhausted and NK cell depletion, which would increase the number of anti-viral
T cells, would not have an impact on viral load as the T cells are exhausted. However, if the viral
burden is lower, then there are still functional T cells that would be spared by NK cell depletion,
which would then mediate viral control (196). In addition to viral persistence, Cook et al. used
an NK cell-depleting antibody to identify a role for NK cells in mediating T cell exhaustion
(324). NK cell depletion prior to Cl13 infection reduced the percentage of transferred P14 T cells
(TCR transgenic CD8 T cells specific for LCMV GP33-41) expressing the inhibitory receptors
PD-1, Lag3 and 2B4, and the per cell expression of these molecules 8 dpi, as well as their
production of IFNγ, TNF and IL-2 following ex vivo re-stimulation on day 29 pi. A role for NK
cells in mediating diminished APC function following Cl13 infection was also identified, in that
APCs isolated from Cl13-infected/NK cell-depleted mice (2-3 dpi) showed an enhanced ability
to stimulate LCMV-specific T cell proliferation ex vivo when compared to APCs isolated from
Cl13 infected/untreated mice (2-3 dpi) (324).
The mechanisms of T cell recognition by NK cells are likely to involve multiple receptor-ligand
interactions. Lang et al. point to a role for NKG2D, in that they found that LCMV infection
upregulates NKG2D and NKG2D ligand expression, and that blocking NKG2D with an antibody
results in viral control, a finding that is similar to NK cell depletion in their model (322). In
contrast, Waggoner et al. found no role for NKG2D in the NK cell-dependent killing of CD4 T
cells, and instead pointed to an interaction between the inhibitory NK cell receptor, 2B4, and its
ligand, CD48 (180, 323). Cl13 infection of 2B4-/-
mice, which have hyperactive NK cells due to
loss of an inhibitory signal, results in reduced T cell responses and a persistent viral infection
that is never cleared (325). The differences in the Lang and Waggoner studies have not been
resolved. They might be due to the differences in the tropism and pathogenicity of the different
LCMV strains used, Cl13 and WE, and differences in the NK ligands upregulated or
31
downregulated on the T cells by the different viruses. It is also possible that different doses of the
depleting NK cell antibody, anti-NK1.1 (200 μg/mouse in the Lang study versus 25 μg/mouse in
the Waggoner study), resulted in depletion of different subsets of NK cells with distinct activities
during LCMV infection.
The T cell-expressed inhibitory NK cell ligands MHC-I and Qa-1b, as well as the ligands for the
activating receptor NCR1, were recently implicated in the mechanism whereby NK cells
recognize and kill virus-specific CD4 and CD8 T cells following LCMV infection. It was shown
that type I IFN signaling early following LCMV infection can protect anti-viral T cells from
perforin-dependent NK cell-mediated cytotoxicity by upregulating NK cell inhibitory ligands
and downmodulating NK cell activating ligands on T cells. LCMV-specific IFNAR1-/-
T cells (T
cells lacking type I IFN receptor 1) were rapidly eliminated following transfer into LCMV-
infected hosts, however, this depletion was abrogated with prior NK cell depletion (326, 327).
NK cells have also been shown to dampen T cell-mediated immunity in other murine viral
infection models, including MCMV (328, 329), Friend virus (FV) (330), and influenza (331).
These functions of NK cells might have evolved to avoid T cell-mediated immunopathology or
development of autoimmunity.
Human genetic studies have shown that the presence of certain combinations of NK cell
inhibitory receptor genes in the KIR family and HLA genes correlates with viral control in
cohorts of HCV-infected individuals (332-334), and a better prognosis in HIV patients (335-
337). Co-expression of the inhibitory KIR3DL1 genotype that exhibits high cell surface
expression (KIR3DL1*h) and its ligand, a HLA-B allele with an isoleucine at position 80 (HLA-
Bw480I
), is associated with slower HIV-1 disease progression (337). Co-expression of the
inhibitory KIR2DL3 and its cognate HLA-C ligand is associated with spontaneous resolution of
viremia following HCV infection and is found more frequently in exposed seronegative aviremic
individuals and in HCV-infected individuals that respond favourably to treatment (332, 333,
338). These inhibitory receptors are thought to be protective by enhancing the activity of NK
cells during these infections. The inhibitory KIR3DL1*h/HLA-Bw480I
pairing that is protective
in HIV infection is believed to be associated with high cell surface expression of the inhibitory
KIR, which leads to strong NK cell licensing, that is, an enhanced functional competence (339).
The inhibitory KIR2DL3/HLA-C1 interaction is thought to be more protective in HCV infection
32
because it mediates a weaker inhibitory signal than other KIR/HLA pairings, thereby being
easily overridden by activating signals (332, 340, 341). One study showed that HIV-exposed
seronegative female sex workers in Abidjan, Côte d’Ivoire, more frequently possessed inhibitory
KIR genes in the absence of their cognate ligand genes, whereas HIV-seropositive workers were
characterized by inhibitory KIR/HLA receptor/ligand pairings (336). Absence of ligands for
inhibitory KIRs was thought to lower the threshold for NK cell activation (336). Indeed, strong
NK cell responses have been associated with inhibition of HIV and HCV replication (332, 342,
343). However, studies of NK cells in LCMV infection suggest that these inhibitory receptors
could also be protective in the human studies because they shut down NK cell responses, which
would otherwise dampen anti-viral immunity and promote viral persistence.
In sum, NK cells were classically known for their role in mediating viral clearance early
following infection, however, mounting evidence from the study of LCMV and other viral
infections suggests that NK cells have an immunoregulatory role that prevents pathology, but
contributes to viral persistence. It seems therefore that depleting NK cells could be a therapeutic
approach in the treatment of chronic viral infections in humans; however, caution must be taken,
as these functions of NK cells presumably evolved to protect the host from immunopathology.
As the Waggoner study showed, depleting NK cells during a high dose Cl13 infection eventually
enhanced immunopathology and mortality (323). More work will be needed before we can
translate these findings into treatments for human disease. These effects of NK cells were largely
shown to be mediated in the first few days following viral infection. In chapter 3 of this thesis, I
identify a novel role for NK cells in the later stages (>3 weeks) of LCMV infection.
1.5.5 Type I Interferons during LCMV infection
Type I IFN production reaches peak titers in the serum 2-3 days pi with ARM (344). Multiple
cell types are induced to produce IFN, including DCs, pDCs, and marginal zone macrophages,
which are thought to be the primary IFN producers (345). IFN expression wanes with the
resolution of ARM infection, but persists following Cl13 infection, and is detectable in DCs out
to day 50 pi (274, 320). The IFNAR-regulated genes, OAS and Mx1, are specifically enriched in
the immunoregulatory APC population expressing high levels of PD-L1 and IL-10 on day 9 post
Cl13 infection, suggesting that prolonged IFN signaling and immunosuppression are linked
(320).
33
IFNs are involved in DC activation and maturation, directly affect T cell
expansion/differentiation, and have also been shown to activate NK cells following LCMV
infection (344, 346-350). Resistance to IFN α/β and γ impacts the ability of various LCMV
strains to spread within the host, in that relatively IFN-resistant strains such as Cl13 spread, but
IFN-sensitive strains such as ARM, do not (351). IFNs can play a critical role in LCMV
infection by reducing viral loads early, and therefore modifying the extent of CD8 T cell
exhaustion and viral persistence, depending on the strain of LCMV (351). Control of ARM
infection certainly depends on a robust IFN response, in that IFNAR-/-
mice show accelerated
kinetics of virus replication, higher peak viral titers, and a slower kinetics of clearance, with
virus still detectable at day 9 pi (320), and variable clearance between 2-8 weeks pi (351).
The IFN signature induced by Cl13 infection, however, can also contribute to
immunosuppression and viral persistence, as alluded to earlier. Cl13 infects hematopoietic
progenitor cells and impairs their development into DCs, a mechanism that is dependent on type
I IFN/STAT2 (76, 274, 352). It was shown that Flt3L treatment of Cl13-infected mice resulted in
reduced expansion of CD8+ and CD8
- DCs compared to uninfected controls, whereas the same
treatment in IFNAR-deficient mice resulted in normal expansion of CD8+ DCs (76). This
strategy evoked by the virus results in fewer DCs available to prime T cells and generate anti-
viral responses. Blocking IFNAR signaling prior to Cl13 infection reduced expression of
molecules associated with immunosuppression (IL-10, PD-L1), but initially increased viral load,
indicating that early IFN signaling was required for viral control. However, when the immune
response was assessed on day 30+, there was now significantly less viremia in the IFNAR
blocking antibody treated mice. Similar results were obtained when IFN signaling was blocked
therapeutically during late stages of infection (day 25+) (320, 353). These and other data suggest
a dual role for IFNs following Cl13 infection - stimulatory early, but immunosuppressive late.
During chronic viral infection in humans, a combined ribavirin/IFN therapy has been shown to
be effective in the treatment of HCV-infected individuals, although some patients do not respond
to this therapy. A characteristic of these patients is a high IFN signature prior to treatment, which
given the findings in Cl13 infection, might mediate immune dysfunction and resistance to any
benefits bestowed by further addition of IFN (354, 355). An elevated interferon signature in
HCV patients can be associated with limited control of virus replication and development of liver
34
pathology (356, 357). In line with these data is the finding that HIV infection of humans and
pathological SIV infection of rhesus macaques is associated with an elevated, prolonged IFN
signature, whereas non-pathogenic SIV infections of sooty mangabeys and African green
monkeys shows curtailed IFN expression (358, 359). Blocking IFN early during SIV infection of
rhesus macaques, however, is not beneficial, in that this was recently shown to reduce anti-viral
genes, increase SIV reservoir size, and accelerate CD4 T cell depletion and progression to AIDS.
In this model, administering IFN-α2 initially prevented systemic infection, but continued
treatment was followed by IFN-desensitization and a worse outcome (360).
Taken together, these data suggest that timing of IFN blockade/treatment is a critical factor in
determining the efficacy of therapy during chronic viral infection in mouse and man. IFNs can be
required early following initial infection to control viral load, therefore blocking IFNs at this
time can be detrimental and administering IFNs beneficial. However, persistent IFN signaling
can induce immune dysfunction, therefore blocking IFNs later during the infection, after initial
viral replication has been controlled, can be beneficial, and administering IFN at this time could
enhance immune dysfunction or have no effect, depending on the functionality of the immune
response. These uncertainties highlight the complexity of targeting type I IFNs therapeutically.
In chapter 3 of this thesis, I investigate the effect of IFN blockade on the population of NK cells
in the spleen and splenic atrophy following Cl13 infection.
1.5.6 Splenic architecture and remodeling following LCMV infection
Adaptive immune responses, such as those that occur following viral infection, are characterized
by substantial restructuring of secondary lymphoid organs, such as the spleen and LNs, including
both their size and architecture. Mesoscopic imaging techniques such as optical projection
tomography (OPT) are suited to probe objects of 0.5 to 15 mm in diameter, which are too large
for conventional microscopic imaging (361, 362). OPT analysis of whole-mount fluorescently
labeled murine peripheral LNs (PLNs) was used to obtain quantitative structural 3D information
on PLN structure following acute LCMV infection (362). This study revealed that reorganization
of PLN microarchitecture following acute LCMV infection, was accompanied by a 3-fold
increase in PLN volume and high endothelial venule network length and required LTα1β2-
expressing B cells, but not VEGF-A (362). During infection, the fibroblastic reticular cell (FRC)
35
network expands by proliferation in order to increase in volume and accommodate more
lymphocytes.
Although immune responses are associated with an increase in the size of lymphoid tissues, their
microarchitecture must be maintained in order to facilitate effective communication between
immune cells, and generate protective T cell and antibody responses. Infection with several
strains of LCMV has been shown to disrupt lymphoid architecture early following infection, in
part due to the CD8 T cell mediated destruction of virally-infected marginal zone macrophages
and FDCs, although organization returns by 3 weeks pi (363). This disrupted architecture is
believed to contribute to the immunosuppressive capabilities of persistent LCMV strains. It was
recently shown that type I IFN production following Cl13 infection also contributes to splenic
disorganization, in that blocking IFN prior to infection prevented disruption of lymphoid tissues
in the spleen (353). Pathological cytokine production following infection with LCMV WE also
contributes to disruption of splenic architecture (364). Blocking CD27 signaling, which was
thought to mediate pathological IFNγ and TNF production by CD4 T cells, abrogated LCMV-
induced immunopathology and actually mediated elimination of an otherwise chronic LCMV
strain, Docile (364). TNF-mediated loss of marginal zone macrophages was also induced by the
parasite Leishmania donovani resulting in lymphoid tissue remodeling and impaired lymphocyte
trafficking into the white pulp (365).
Cl13 can also infect and disrupt the conduit function of FRC, a network of cells which provides a
3-dimensional framework in SLOs on which immune cells travel, interact, and receive survival
signals (366). By immortalizing FRC in vitro, it was shown that these cells also present antigen
in an MHC-I/II-dependent manner and provide activation signals to LCMV-specific CD4 and
CD8 T cells (367). The disrupted function of these cells and their expression of PD-L1 following
Cl13 infection are thought to contribute to immunosuppression and viral persistence (366, 368).
Damage to the FRC following HIV infection is also thought to contribute to progressive
depletion of CD4 and CD8 T cell populations, as these cells are prevented from receiving
survival factors such as IL-7 through the FRC network (369).
Restoration of lymphoid tissue architecture following LCMV infection is mediated in part by
adult LTi. Following LCMV WE infection, LTi proliferate and interact with stromal cells in a
LTβR-LTα1β2-dependent manner, thereby restoring lymphoid organ structure following infection
36
(370). Thus, similar to their role in ontogeny, LTi are also involved in maintaining the structural
integrity of lymphoid tissues later in life. Embryonic mesenchymal progenitors of the Nkx2-
5+Islet1
+ lineage, which give rise to embryonic mesenchymal cells with lymphoid tissue
organizer activity, were also recently shown to be involved in regenerating FRC and stromal cell
integrity following LCMV WE infection. Although the precise nature of the cells that participate
in the regeneration of stromal networks in the spleen is not known, these authors point to a
potential role of perivascular cells, which were recently suggested to function as mesenchymal
stem cells. These cells are not targeted by LCMV, thereby making them candidates for local
stromal progenitors during tissue regeneration (213).
Remodeling of splenic architecture following infection also occurs with other viruses such as
MCMV. MCMV infection leads to loss of marginal zone macrophages, and specifically
downregulates CCL21 expression by splenic stromal cells in an LT-independent manner,
resulting in aberrant T cell localization within the white pulp (371). This is thought to be a
strategy used by the virus to impede cellular trafficking and cell-cell interactions required to
mount effective immune responses.
In sum, the destruction of lymphoid architecture required to mount anti-LCMV immune
responses is another reason that LCMV continues to replicate and persist throughout the host.
This is a reversible process, in that splenic architecture is eventually regained; however, the
mechanisms underlying the regeneration of lymphoid tissue integrity are just beginning to be
elucidated.
1.5.7 Resolution of inflammation following LCMV infection
Although the expansion phase of the immune response (0-8 dpi) following LCMV infection has
been well characterized, less is known about the resolution of inflammation during the
contraction phase (day 8+). The loss of cell numbers is largely attributed to the apoptosis of CD8
T cells, due to the unavailability of survival cytokines in the absence of viral replication, activity
of multiple death-inducing TNF family receptors, and toxicity due to repeated release of perforin
and IFNγ (372-375). The rate of CD8 T cell contraction following ARM infection is independent
of the dose and duration of infection, the magnitude of expansion, or the amount of antigen
displayed (376). Following clearance of infectious virus, CD8 T cells no longer proliferate;
37
however, CD4 T cells show delayed contraction compared to CD8 T cells, due to their continual
proliferation in response to residual antigen (305, 377-381).
The prototypical member of the death-inducing TNFR family is TNFR1. ARM infection of
TNFR1-deficient mice revealed that this molecule is not a critical player in the downregulation
of CD8 T cell responses; however, delayed contraction is observed in TNFR1/2 DKO mice (382-
384). TNF deficiency prolonged the effector phase of the CD8 T cell response and increased the
number of memory CD8 T cells following ARM infection due to reduced apoptosis (385).
TNFR1 deficiency alone and in combination with TNFR2 deficiency delayed contraction of CD8
T cells specific for GP33-41 and GP276-286 following Cl13 infection (386). Similar to TNFR1, the
TNFR family member Fas is largely dispensable for the apoptosis of LCMV-specific CD8 T
cells (387, 388); however, combined Fas/BIM deficiency leads to a LN-specific block in
contraction following ARM infection (389). BIM was also shown to play a critical role in the
downregulation of CD8 T cell responses following Cl13 infection, in that BIM deficiency
prevented the apoptosis of NP396-404-specific CD8 T cells (390). Taken together, these data
suggest that the strain of LCMV, as well as the specificity of the CD8 T cells differentially
impact the genes and pathways utilized in the contraction phase of the immune response. How
other cell types are depleted during the contraction phase is not well understood, and is the topic
of chapter 3.
1.6 Murine models of viral infection: influenza
1.6.1 Influenza virus
Pandemic influenza infection of humans claimed between 20 and 50 million lives in the 20th
century, and today seasonal influenza virus remains a significant cause of morbidity and
mortality especially in the immunocompromised, young children, and the elderly (391). The
enveloped influenza virus is a member of the orthomyxoviridae family and contains a genome
composed of 8 segments of negative sense ssRNA surrounded by NP. The major targets of the
anti-influenza antibody response are the envelope glycoproteins hemagglutinin (HA) and
neuraminidase (NA), of which there are several subtypes. HA mediates binding of the virus to
sialic acid residues on target cells and fusion of the viral envelope with the endosome membrane
whereas NA allows the virus to be released from the host cell by cleavage of the sialic acid
38
groups from glycoproteins (392). Due to the error prone polymerase, the influenza virus
continuously mutates, and viral variants with mutations in HA that abrogate recognition by
neutralizing antibodies are selected in a process termed antigenic drift. Antigenic drift results in
seasonal influenza viruses that require new vaccines. Antigenic shift, the process by which RNA
segments of two viruses reassort, however, can result in novel HA or NA subtypes to which
humans have little or no immunity, potentially resulting in a pandemic.
1.6.2 Innate immune responses
Influenza infection of mice is used as an animal model to study influenza pathogenesis. Influenza
A/HKx31 (X31), an H3N2 virus, and the more virulent A/PR8 (PR8), an H1N1 virus, are
commonly used strains in the laboratory. X31 elicits a mild infection that is cleared within 8
days, whereas PR8 infection results in a more severe disease outcome that can culminate in
death. X31 is a recombinant virus consisting of the H and N segments of the Hong Kong 1968
virus, with the other genes of the PR8 virus, thus the two viruses share common CD8 T cell
epitopes (393, 394). Animal models have shown that influenza infection of epithelial cells in the
respiratory tract is first detected by the innate arm of the immune system. Innate receptors such
as RIG-I and NLRP3 mediate cell-intrinsic recognition by detecting virus that is present within
the cytosol of infected cells. TLRs such as TLR3, which detects dsRNA in virus-infected cells,
and TLR7, which detects viral ssRNA that has been taken up into endosomes of infected cells
mediate cell-extrinsic recognition. Viral recognition leads to the secretion of type I IFNs, pro-
inflammatory cytokines and chemokines such as IL-1, IL-6 and TNF, eicosanoids, and the
upregulation of adhesion molecules required for leukocyte migration. Type I IFNs, produced by
macrophages, pneumocytes, DCs and pDCs, induce an anti-viral state through the expression of
IFN-stimulated genes (ISGs), such as MX proteins, IFITM proteins and PKR, in neighbouring
cells (395).
The release of chemokines leads to the recruitment of other cell types such as neutrophils,
monocytes, NK cells, and memory T cells (396). NK cells produce large amounts of IFNγ and
have been shown to kill influenza-infected epithelial cells. Mice lacking the activating NCR1
receptor on NK cells were shown to have a higher mortality rate following influenza infection
(184). Monocytes, neutrophils and alveolar macrophages help clear infected dead cells, which is
an important mechanism involved in viral clearance (397). Respiratory DCs (RDCs) are also
39
major players in the innate immune response to influenza infection. Since influenza infection is
mainly restricted to the respiratory tract, it is the antigen bearing RDCs migrating to the LNs that
initiates adaptive immunity in lymphoid tissues. As early as 6 hours following infection, these
DCs upregulate costimulatory molecules, MHC-II, adhesion molecules, and CCR7, in order to
migrate to the LNs and activate influenza-specific CD4 and CD8 T cells (398-402). The CD103+
RDC subset has been identified as the main APC in the initial priming of T cells, after which the
CD11bhi
DCs continue presenting antigen in the LNs. The former subset of DCs efficiently
differentiates CD8 T cells that are associated with migration to the lung, whereas the latter subset
differentiates CD8 T cells that are associated with retention in the LNs (403, 404). DCs in the
lung have also been shown to be important in maintaining CD8 T cell survival in the lung (405).
1.6.3 Adaptive immune responses
Within 5-7 days pi, antigen-specific CD4 and CD8 T cells home to the lung. Depending on the
target cell encountered, CD8 T cells kill infected cells in a Fas/FasL or perforin/granzyme
dependent manner and/or produce inflammatory cytokines such as TNF and IFNγ (394).
Antigen-specific CD4 T cells are important in the production of anti-influenza antibodies by B
cells, but also produce anti-viral cytokines, can kill virally infected cells, and play a role in the
development of anti-influenza CD8 T cell responses (406-409).
Although the immune response generated against influenza infection is meant to clear the virus
and protect the host, it can also cause severe immunopathology leading to morbidity or mortality
of the host. Activated CD4 and CD8 T cells, as well as macrophages secreting nitric oxide and
oxygen radicals can mediate lung tissue damage (391, 410, 411). Therefore, as with LCMV
infection, the immune system must strike a balance – clear the virus, but spare the host.
Costimulatory members of the TNFR family, such as 4-1BB, GITR, OX40 and CD27, have been
shown to impact multiple stages of the immune response against influenza infection, including
the priming of antigen-specific T cells by DCs, effector and memory T cell proliferation,
survival, and effector function, as well as the development of memory and secondary response to
influenza (412-414). The TNFR family, and specifically, 4-1BB and 4-1BBL, will be further
discussed in the next section.
1.7 TNF/TNFR family
40
1.7.1 Overview
TNF, the founding member of the TNF superfamily, earned its name following the initial
observations that it could mediate tumor regression (415). The TNF superfamily is now known
to include 30 receptors in the TNFR family and 19 ligands in the TNF family (416). The success
of TNF blockers such as Etanercept (Enbrel; Amgen/Pfizer) in several inflammatory conditions
has garnered much interest in harnessing the immune modifying capabilities of the other
members of this family for therapeutic benefit (417). Reagents that either stimulate or block the
activity of other TNF superfamily members are being evaluated in clinical trials, and a few have
been approved for human use to treat autoimmune/inflammatory conditions and cancer (417).
For example, a depleting anti-CD30 specific antibody has been approved for treatment of
Hodgkin’s lymphoma and anaplastic large-cell lymphoma and an antagonizing human BAFF-
specific antibody has been approved for treatment of SLE (417). TNFR family members are type
I transmembrane proteins, characterized by cysteine-rich extracellular domains, although
following cleavage, they can also exist in soluble form. TNF family members include type II
transmembrane proteins that are also found in both membrane-bound and soluble form, and
similar to their respective receptors, are thought to associate and mediate their effector functions
as trimers. It is the cytoplasmic portion of the TNFRs that broadly divide this family into two
groups: pro-survival and pro-death (415). Members that contain a death domain (DD), such as
Fas, associate with DD-containing proteins to signal apoptosis, whereas members without DDs,
such as 4-1BB, GITR, CD27 and OX40 can associate with TNFR associated factors (TRAFs) to
mediate survival signaling (413). Of course, this simple dichotomy is much more complex, in
that some pro-death members can mediate survival signals under certain conditions, and vice
versa. Here, I will discuss the TNFR family member 4-1BB and its ligand 4-1BBL, which are the
focus of chapter 2.
1.7.2 4-1BB/4-1BBL
Of the TNFR family, 4-1BB (CD137, TNFRSF9) is especially interesting due to the ability of
anti-4-1BB antibodies to potently enhance T cell responses in the context of cancer and viral
infections (418, 419), and induce immunoregulatory activity in models of autoimmunity and
inflammatory disease (413, 420). Although these antibodies have been associated with cell
toxicity (421-423), modifying the dose and timing of administration can reduce toxic side
41
effects. Several antibodies to 4-1BB are currently being evaluated in clinical trials for cancer
(417, 424, 425).
4-1BB and its ligand, 4-1BBL, are largely pro-survival costimulatory members of the
TNFR/TNF family. Mouse 4-1BB, a type I transmembrane protein, was initially discovered and
cloned in 1989 by screening cDNA that was selectively expressed in activated T cells (426),
followed by the identification of mouse 4-1BBL, a type II transmembrane protein, by Goodwin’s
group in 1993 (427). Human 4-1BB and 4-1BBL were subsequently characterized by other
groups (428-430). The 4-1BB gene is located on chromosome 4 in mice and chromosome 1 in
humans, and the 4-1BBL gene on chromosome 17 in mice and chromosome 9 in humans.
Similar to other transmembrane proteins of the TNFR/TNF family, 4-1BB and 4-1BBL mediate
their activity by associating as trimers (431).
Much research activity has focused on the role of 4-1BB/4-1BBL on multiple subsets of T cells
in anti-viral and anti-tumor immunity, where this pathway has been shown to provide a CD28-
independent costimulatory signal, induce survival signaling via upregulation of survival
molecules such as Bcl-xL and Bfl-1 and downregulation of pro-apoptotic molecules such as BIM,
and enhance cell cycle progression and cytokine production (413, 414, 432, 433). 4-1BB is not
expressed on naive T cells, but NF-κB and MAPK signaling can upregulate 4-1BB expression on
both CD4 and CD8 T cells (413). 4-1BB expression on these cells is very rapid and transient in
vivo, but the duration of 4-1BB expression can be prolonged under conditions were antigen
persists (434-438). 4-1BBL is predominantly expressed on APCs, where it can be regulated by
CD40, IgM, and TLR signaling in vitro (439-441). 4-1BBL expression is also detectable on
hematopoietic progenitors cells and at sites of inflammation on cardiac myocytes, neurons, and
aortic tissue (432).
1.7.3 Role of 4-1BB/4-1BBL on T cells
The study of 4-1BB- and 4-1BBL-deficient mice and the use of agonistic anti-4-1BB antibodies
have highlighted the importance of this pathway in enhancing immunity against viral infections,
such as influenza, LCMV, VSV, VV, FV, MCMV, and MHV68 (413, 414, 432, 442). When 4-
1BBL-/-
mice were infected with influenza X31, it was shown that 4-1BBL was not required for
the primary influenza-specific CD8 T cell response, but for sustaining the number of memory
42
CD8, but not CD4, T cells and the recall response to influenza (443). Recent data show that
GITR controls the antigen-independent expression of 4-1BB on memory CD8 T cells in the BM
and liver (444), and that 4-1BB-expressing memory CD8 T cells plausibly receive survival
signals from 4-1BBL expressing stromal cells in the BM (445).
In contrast to these studies, a role for 4-1BB/4-1BBL has been identified in the primary T cell
response against viral infection in other models (436, 446-448). Whether this pathway influences
the primary response seems to depend on the severity of infection and persistence of the virus.
For example, during mild respiratory influenza infection with X31, 4-1BB is only transiently
upregulated on lung CD8 T cells and is dispensable for the magnitude of the primary CD8 T cell
response and mouse survival. However, during severe respiratory influenza infection with the
PR8 strain, 4-1BB expression on lung CD8 T cells is sustained, and critical for CD8 T cell
accumulation in the lungs, viral clearance and mouse survival. In this model, 4-1BB acts to keep
the CD8 T cells alive long enough to clear the infection (436). When virus cannot be cleared,
such as in chronic infection with LCMV clone 13, the 4-1BB pathway in CD8 T cells becomes
desensitized due to loss of the signaling adapter TRAF1 (437).
CD4 T cell responses are also modulated by 4-1BB/4-1BBL signaling, however, most studies on
the role of 4-1BB/4-1BBL in anti-viral immunity have identified a role for this pathway
predominantly in CD8 T cells. Indeed, in some models, 4-1BB is induced at a higher level on
CD8 compared to CD4 T cells (449, 450). The role of endogenous 4-1BB/4-1BBL seems to be
mainly in enhancing CD8 T cell survival (436, 443, 451), however, 4-1BB stimulation has also
been shown to modulate T cell proliferation, cytokine production and effector function (413).
Tregs represent a double-edged sword in viral immunity, in that their activities can suppress host
immunity to viral infection and enable viral persistence; however, they can also be deemed
beneficial when dampening immune responses associated with immunopathology (452, 453). In
contrast to naive T cells, where 4-1BB is upregulated upon activation, 4-1BB is constitutively
expressed on Tregs and can be increased by TCR stimulation and IL-2 treatment (454-457).
Anti-4-1BB costimulation has been shown to render effector CD8 T cells resistant to virus-
induced Treg-mediated suppression (458); however, in some models, it is possible that the
effects of 4-1BB are directly on Tregs, as this pathway has been shown to modulate Treg
43
expansion. However, there are conflicting data on whether 4-1BB on Tregs enhances or
abrogates their suppressive function (454-456, 459).
1.7.4 Role of 4-1BB/4-1BBL on non-T cells
The effects of 4-1BB and 4-1BBL in the aforementioned studies and in the vast majority of
studies have been attributed to the role this pathway plays in T cell biology, however 4-1BB is
broadly expressed on other cell types, including APCs (441, 460-465), NK/NKT cells (466-469),
hematopoietic progenitor cells (470), mast cells (471), and neutrophils (472, 473). How 4-1BB
expression on these cell types is integrated with 4-1BB expression on T cell subsets is not
understood. As this thesis explores the role of 4-1BB and 4-1BBL on DCs, these cells will be
discussed in more detail.
1.7.5 Role of 4-1BB/4-1BBL on DCs
Several stimuli, including LPS, anti-CD40, TNF, IFNγ, IL-12, Zymosan, and GM-CSF can
induce or upregulate 4-1BB expression on DCs either in vitro or ex vivo (441, 460-462), and
although endogenous 4-1BB appears to be dispensable for DC maturation (460), it has been
shown to modulate DC function. Anti-4-1BB treatment of DCs was shown to induce cytokine
(IL-6 and IL-12) production (441, 462), survival molecules (Bcl-xl and Bcl-2) (460, 464), and an
enhanced ability to stimulate T cell proliferation, independent of effects of T cells (462).
However, 4-1BB on DCs has also been suggested to suppress immunity. Anti-4-1BB treatment
at the beginning of LCMV Armstrong infection resulted in activation-induced cell death (AICD)
of CD8 T cells and immunosuppression, whereas delaying the treatment by 3 days enhanced
virus-specific CD8 T cell responses and mediated rapid viral clearance (474). In a follow-up
report, the deleterious effects of early anti-4-1BB treatment were argued to involve targeting of
both T cells and DCs. Cotransfer experiments of virus-specific T cells and other lineages into
WT and 4-1BB-/-
hosts followed by LCMV infection and anti-4-1BB treatment showed that only
cotransfer with DCs mediated AICD of virus-specific CD8 T cells. In vitro, anti-4-1BB
treatment of DCs was shown to mediate STAT3 activation, which has been shown to enhance the
ability of DCs to induce antigen-specific T cell tolerance. The authors proposed that early
signaling by 4-1BB on DCs might be involved in the early events that program T cell contraction
following viral infection (463). In another model supporting a regulatory role for 4-1BB on DCs,
44
it was shown that anti-4-1BB treatment of mesenteric LN DCs induces RALDH activity and
promotes iTreg development (461).
The above studies reveal the discrepancy in the literature on whether 4-1BB signaling on DCs
enhances immune responses or downmodulates them. The effects of anti-4-1BB on DC activity
are probably model-dependent as timing of administration and cell location were different in the
above studies. It is also not clear what endogenous 4-1BB does on DCs, as the handful of studies
that have looked at the function of 4-1BB on DCs have mainly tested the effects of
supraphysiological signaling of 4-1BB by use of anti-4-1BB antibodies or 4-1BBL-
overexpressing cell lines. Moreover, the DC-intrinsic roles of 4-1BB/4-1BBL molecules during
an ongoing immune response in vivo are not known. In chapter 2 of this thesis, I explore the
endogenous function of 4-1BB and 4-1BBL on BM-derived DCs.
1.7.6 4-1BB signaling
The cytoplasmic tails of 4-1BB and other TNFRs do not possess intrinsic enzymatic activity, and
therefore associate both directly and indirectly with a family of adapter proteins, TRAFs, to
mediate intracellular signaling. This association is directly mediated via TRAF-interacting motifs
consisting of 4-6 amino acids in the cytoplasmic tail (475-477). Although initially identified
downstream of TNFR2 (478) and other members of the TNFR family, it is now well appreciated
that TRAFs effect multiple receptor signaling pathways, including TLRs, NLRs, and TCRs, and
that their aberrant regulation is associated with several human diseases (479). To date, there are 6
identified mammalian TRAFs: TRAF1-6. All members contain a conserved C-terminal TRAF
domain, which consists of an N-terminal coiled-coil region (TRAF-N) and a C-terminal-β-
sandwich (TRAF-C), involved in protein-protein interactions with receptors, TRAFs, and other
signaling molecules (479). Minor structural differences in the TRAF-C domain accounts for
binding to different receptors (480, 481), whereas the TRAF-N region is responsible for
homotypic and heterotypic interactions with other TRAF molecules (479). Apart from TRAF1,
all members also contain an N-terminal RING domain that can potentially mediate E3 ubiqutin
ligase activity (479).
Mouse 4-1BB recruits TRAFs 1 and 2, and mediates both classical and alternative NF-κB
activation, as well as extracellular signal-regulated kinase (ERK)/c-Jun N-terminal kinase
45
(JNK)/p38 MAPK and delayed PI3K/AKT activation (Fig. 1.2) (414, 482-485). TRAF1 has been
shown to be required for ERK activation-dependent BIM downmodulation downstream of 4-
1BB in T cells (486). Recent results show that the F-actin binding protein LSP-1 is recruited to
the 4-1BB signalosome via TRAF1 and that in the absence of LSP-1, T cells fail to activate ERK
during 4-1BB signaling (487). 4-1BB can activate the alternative NF-κB pathway via TRAF3
degradation, with delayed kinetics compared to activation of the classical NF-κB pathway (485).
TRAF1 was found to restrain costimulation-independent activation of the alternative NF-κB
pathway in T cells, whereas it is essential for early activation of the classical NF-κB pathway and
survival signaling downstream of 4-1BB in T cells (485, 488). In resting T cells, the activation
of the alternative NF-kB pathway is restricted by the E3 ligase activity of cellular inhibitor of
apoptosis (cIAP) 1 and 2, which are brought into proximity with the alternative NF-κB activating
kinase, NIK, through association with TRAF2 (489, 490).
Of interest, Zheng et al. (491) have shown that a heterotrimer of two TRAF2 with one TRAF1
coiled coil region preferentially and asymetrically recruits a single cIAP BIR domain, compared
to a TRAF2 homotrimer. The TRAF1 (TRAF2)2 N domain heterotrimer more efficiently recruits
cIAPs, thus this in turn may allow TRAF2 to more efficiently recruit cIAPs to act as E3 ligases
for NIK degradation in anti-CD3-activated cells than a TRAF2 homotrimer. However, as TRAF1
has limited tissue distribution (492), it clearly isn’t required to restrict NIK in all cells. Thus,
McPherson and colleagues suggested that the fact that TCR stimulation increases the levels of
the NIK substrate, P100, in cells, may be the reason why TRAF1 is needed in addition to TRAF2
to efficiently recruit cIAP molecules (485). Alternatively, TRAF1 is also needed to protect
TRAF2 from degradation downstream of 4-1BB (486) and other TNFR family members (493,
494). Thus, the role of TRAF1 in preventing constitutive NIK activation could be due to it
stabilizing TRAF2 against degradation in activated T cells. Further work will be required to
resolve this issue. Interestingly, in the context of chronic viral infection in mouse and man,
TRAF1 is specifically lost from chronically stimulated PD-1hi
virus-specific CD8 T cells,
thereby desensitizing the 4-1BB pathway (437). TRAF1 degradation was shown to be mediated
at least in part by a TGFβ-dependent mechanism. This effect required protein synthesis
downstream of TGFβ and was sensitive to chloroquine, suggesting that a TGFβ-induced gene
promotes TRAF1 degradation in chronically stimulated T cells, perhaps via autophagy, although
this requires further study.
46
This figure was modified and reproduced from (414)
47
4-1BB has recently been shown to interact with Galectin-9, a member of the β-galactoside-
binding family of lectins, at sites in the CDR4 that are independent of the binding sites for anti-4-
1BB or 4-1BBL. This interaction, which is thought to involve 4-1BB and galectin 9 on the same
cell, may facilitate the aggreggation of 4-1BB monomers, dimers, or trimers when ligated by
anti-4-1BB or 4-1BBL, thus mediating productive signaling. In the absence of Galectin-9, the
stimulatory function of anti-4-1BB in in vivo models, and in the in vitro stimulation of T cells,
DCs, and NK cells is impaired (495).
1.8 Thesis Rationale
As outlined in this introductory chapter, innate immune responses orchestrated in secondary
lymphoid organs, such as the spleen, play a key role in modulating the outcome of viral
infection. My original goal was to study the role of 4-1BB and 4-1BBL on innate immune cells,
and how this impacts anti-viral immunity. Chapter 2 asked about the expression and function of
4-1BB/4-1BBL on DCs, and whether these molecules intrinsically modulate the ability of DCs to
generate influenza-specific T cell responses in the spleen. Chapter 3 emerged from a study of the
role of the 41BB signaling pathway during ARM and Cl13 infection, where it was noted that 60
days following Cl13 infection, the spleen remained small even weeks after LCMV was no longer
detectable in the spleen. I went on to analyze this process in-depth and uncovered a role for NK
cells in viral-induced splenic atrophy.
48
Chapter 2
4-1BB and 4-1BBL constitutively interact on LPS-activated dendritic cells but
dendritic cell-intrinsic 4-1BB/4-1BBL interactions are dispensable for
cytokine production and anti-viral CD8 T cell priming
Mbanwi AN, Lin GH, Sabbagh L, Watts TH. 4-1BB and 4-1BBL constitutively interact on LPS-
activated dendritic cells but dendritic cell-intrinsic 4-1BB/4-1BBL interactions are dispensable
for cytokine production and anti-viral CD8 T cell priming.
Manuscript in preparation.
Author contributions
Mbanwi AN and THW designed the experiments and wrote the paper.
Mbanwi AN performed all the experiments.
Lin GH performed the initial experiments showing that the anti-4-1BBL antibody TKS-1 only
detects 4-1BBL in the absence of 4-1BB on LPS-activated bone marrow-derived DCs.
Sabbagh L gave technical advice on how to clone the pLJM1-4-1BBL-citrine construct and
transduce cell lines and DCs.
49
2.1 Abstract
Stimulation of the TNFR family member 4-1BB has been shown to potently activate T cell
responses in models of viral infection and cancer. However, 4-1BB is widely expressed on other
cells of the immune system, including dendritic cells (DCs), which also express its ligand, 4-
1BBL. The precise nature of 4-1BB/4-1BBL expression on DCs is not known and the
endogenous function of 4-1BB/4-1BBL on this cell type has remained largely elusive. Here, we
show that LPS stimulation induced both 4-1BB and 4-1BBL expression on bone marrow-derived
DCs, and that these molecules constitutively interact on LPS-activated DCs. However, this
interaction was shown to be dispensable for DC survival, the upregulation of key costimulatory
molecules, cytokine/chemokine production and the ability to stimulate T cell proliferation in
vitro following LPS stimulation. Using an in vivo DC vaccination approach, we further show that
DC-intrinsic 4-1BB and 4-1BBL do not modulate the ability of DCs to induce clonal expansion
of influenza-specific CD8 T cells. The finding of constitutive interaction between 4-1BB and 4-
1BBL on DCs suggests a role for these molecules in regulating DC function; however, the
precise nature of this regulation remains to be elucidated.
2.2 Introduction
The tumor necrosis factor receptor (TNFR) family member 4-1BB was originally identified as an
inducible molecule on activated T cells (426, 496). Its TNF family member ligand, 4-1BBL, is
mainly expressed on activated antigen-presenting cells (APCs) (427, 441), but has also been
detected on hematopoietic progenitor cells (470, 497, 498). 4-1BB signaling in vitro has been
shown to enhance both CD4 and CD8 T cell activation (413, 499), although in vivo, the pro-
survival effects of 4-1BB are more prominent on CD8 compared to CD4 T cells (443, 451, 486,
500). The anti-tumor and anti-viral effects of 4-1BB agonistic antibodies and 4-1BBL vectors
have been largely attributed to stimulation of 4-1BB on T cells (445, 501, 502), however,
functional 4-1BB is expressed on other cells of the immune system, including DCs (441, 460-
464), NK cells (467, 468), NKT cells (466, 469), monocytes (429), neutrophils (472, 473), B
cells (465) and mast cells (471), although the direct effects of 4-1BB on non-T cells in vivo has
not been rigorously studied.
50
DCs are key sentinels of the immune system that orchestrate the T cell response via their ability
to present antigens and costimulatory molecules to T cells, as well as by their production of
chemokines and cytokines (50, 503). Several TNFRs including CD40, RANK and LTβR have
been shown to play a role in DC biology (504). 4-1BB has also been reported on DCs (441, 460-
462), yet its activity on this cell type has not been extensively studied. In addition to 4-1BB, 4-
1BBL has been reported to be induced on activated DCs (441), raising the question of whether
intrinsic 4-1BB/4-1BBL interactions regulate DC function. In this report, we investigate the
expression of 4-1BB and 4-1BBL on bone marrow-derived DCs and provide evidence that 4-
1BB and its ligand can constitutively interact on DCs. However, despite the evidence of a
constitutive interaction, we find that 4-1BB/4-1BBL interactions are not required for LPS-
induced upregulation of costimulatory molecules, DC survival, cytokine/chemokine production,
nor the ability of DCs to stimulate T cell proliferation in vitro. Using an in vivo DC vaccination
approach, we also show that DC-intrinsic 4-1BB and 4-1BBL are dispensable for the primary
clonal expansion of influenza-specific CD8 T cells.
2.3 Materials and Methods
2.3.1 Mice
C57BL/6 wild-type (WT) mice were purchased from Charles River Laboratories (Saint-
Constant, QC, Canada). CD45.1 congenic mice and OT-I TCR transgenic mice were obtained
from The Jackson Laboratory (Bar Harbor, ME, USA). 4-1BB-/-
mice extensively backcrossed to
the C57BL/6 background were bred in our facility. These mice were previously provided to us
by Dr. Byoung S. Kwon (National Cancer Center, Ilsan, Korea). 4-1BBL-/-
mice were originally
obtained under a materials transfer agreement from Immunex (Amgen, Thousand Oaks, CA,
USA) and further backcrossed to the C57BL/6 background in our facility. 4-1BB-/-
and 4-1BBL-/-
mice were crossed to generate 4-1BB-/-
4-1BBL-/-
(DKO) mice. Heterozygotes from a C57BL/6
and 4-1BBL-/-
backcross were further crossed to generate WT and 4-1BBL-/-
littermate controls.
Littermate controls were selected either from the same mother or sisters from the same harem
cage. Littermate controls were used when indicated in the figure legends. Mice were maintained
under specific pathogen-free conditions in sterile microisolators at the University of Toronto. All
51
mouse experiments were approved by the University of Toronto animal care committee in
accordance with the regulations of the Canadian Council on Animal Care.
2.3.2 DC cultures and surface markers
Femurs and tibias were removed from mice (WT, 4-1BB-/-
, 4-1BBL-/-
, and DKO) and bone
marrow cells were harvested by flushing with PBS. Bone marrow cells were seeded at 5 × 105
cells/ml in 100 mm bacteriological Petri dishes. RPMI-1640 media supplemented with 10% heat-
inactivated fetal calf serum, 1X NEAA, 1X GPPS, 50 μM 2-mercaptoethanol, and 40 ng/ml GM-
CSF was used to culture DCs. Fresh media was added on day 3, and on day 6, one half of the old
media was replaced with new media. Nonadherent cells were harvested on days 7-10 for
analysis. DC cultures were always used at a final concentration of 1 × 106
cells/ml. To activate
DCs, immature DCs were stimulated with either 10 ng/ml or 1 μg/ml LPS (026:B6; Sigma-
Aldrich) in 24-well plates or 15 ml round-bottom polypropylene tubes.
Prior to detection of surface markers, WT, 4-1BB-/-
, 4-1BBL-/-
and DKO DCs were treated with
anti-CD16/CD32 Fc block (clone 93) for 15 minutes on ice to prevent antibodies from binding to
the Fc receptors. DCs were then surface stained with a viability dye [LIVE/DEAD Violet
Viability/Vitality Kit (Life Technologies)], and the following antibodies: biotinylated anti-MHC-
I (clone AF6-88.5.5.3), MHC-II-FITC (clone Y3P), CD11c-APC (clone N418), biotinylated
CD80 (clone 16-10A1), biotinylated CD86 (clone GL1), biotinylated CD40 (clone 1C10),
CD45.1-FITC (clone A20), CD45.2-FITC (clone 104), biotinylated 4-1BB (clone 3H3),
biotinylated 4-1BBL (clone TKS-1), biotinylated 4-1BBL (clone 19H3), streptavidin-PE, and
streptavidin-Alexa Fluor 488. Antibodies were either purchased from eBioscience (MHC-I,
CD11c, CD45.1, CD45.2, streptavidin-PE, streptavidin-Alexa Fluor 488) or purified from
hybridomas in our laboratory (CD80, CD86, CD40, MHC-II, 4-1BB, 4-1BBL). Fluorescence
minus one (FMO) controls, 4-1BB-/-
and 4-1BBL-/-
mice were used as staining controls. Surface
marker expression was assessed by flow cytometry.
2.3.3 Blocking studies
293T cells overexpressing 4-1BBL (pLJM1-4-1BBL-citrine) or LPS-activated 4-1BB-/-
DCs
were pre-treated with either PBS, 10 μg/ml 4-1BB-alkaline phosphatase (4-1BB-AP) (439), 10
μg/ml anti-4-1BBL (clone TKS-1), 10 μg/ml anti-4-1BBL (clone 19H3), 10 μg/ml AP, or 10
52
μg/ml Rat IgG for 10 minutes on ice. Cells were then surface stained with anti-CD11c-APC
and/or either biotinylated anti-4-1BBL (clone TKS-1) or biotinylated anti-4-1BBL (clone 19H3)
followed by secondary streptavidin-PE. FMO controls and/or 4-1BBL-/-
DCs were used as
staining controls. Surface marker expression was assessed by flow cytometry.
2.3.4 DC survival assay
Immature DCs (WT and DKO) were stimulated with 1 μg/ml LPS and their survival rate was
tracked on day 0 prior to LPS treatment and every day for 5 days following LPS treatment. DCs
were surface stained with anti-CD16/CD32 Fc block, a viability dye and antibodies to CD11c
and MHC-II, as described above. Following surface staining, DCs were fixed and permeabilized
using Cytofix/Cytoperm (BD Biosciences), and stained intracellularly with anti-Bcl-xL-Alexa
Fluor
488 (clone 54H6; Cell Signaling Technology) and anti-Bcl-2-FITC (clone 10C4;
eBioscience). DCs were then analyzed by flow cytometry.
2.3.5 Cytokine production
Supernatants were first collected from immature DCs (WT and DKO/4-1BBL-/-
) and stored at -
80°C. These DCs were then stimulated with 1 μg/ml LPS. Supernatants were collected 7, 24, 48
and 72 hours following LPS treatment and stored at -80°C. Thawed supernatants were
appropriately diluted and analyzed for the presence of mouse IL-6, IL-12, IL-18, MCP-1 and
RANTES using a custom FlowCytomix multiplex kit (eBioscience) as per the manufacturer’s
instructions. The presence of IL-2 and TNF in the thawed supernatants was determined using
ELISA kits (mouse TNF-α and mouse IL-2 ELISA Ready-SET-Go!) as per the manufacturer’s
instructions.
2.3.6 In vitro antigen uptake and presentation
Immature DCs were treated with 10, 25, 50 or 100 MOI of replication defective adenovirus 5
(Ad)-SIINFEKL-GFP [a gift from Dr. Jonathan Bramson (McMaster University, Hamilton, ON,
Canada)], or 1, 10, or 50 MOI of Ad-OVA, or various concentrations of SIINFEKL peptide and
stimulated with 1 μg/ml LPS. Adenoviruses were added to DC cultures in 15 ml round-bottom
polypropylene tubes. Tubes were then centrifuged at 2200 rpm at 37°C for 2 hours. After 24 and
48 hours, GFP-transduced DCs were washed and stained with a viability dye and antibodies to
53
CD11c and MHC-II and analyzed by flow cytometry. Ad-OVA transduced DCs were washed
and stained with a viability dye and antibodies to CD11c, MHC-II and SIINFEKL-H-2Kb (clone
25-D1.16; eBioscience) or mixed with CFSE-labelled OT-I CD8 T cells at a ratio of 104 DCs to
105
OT-I CD8 T cells. SIINFEKL-loaded DCs were also cocultured with CFSE-labelled OT-I
CD8 T cells. CFSE dilution 3-4 days following DC-T coculture was analyzed by flow cytometry.
To generate CFSE-labelled T cells, CD8 T cells were first purified from spleens of naive OT-I
mice using a negative selection mouse CD8 T cell enrichment kit (STEMCELL Technologies).
Purified CD8 T cells were then stained with 1 μM CFSE for 10 minutes at 37°C and washed to
remove excess CFSE.
2.3.7 DC vaccination
Ad-NP expresses influenza NP in the E1 region and was kindly provided by Dr. Jonathan
Bramson at McMaster University, Hamilton, ON, Canada. The NP was derived from influenza
A/PR8 GenBank: J02147.1. 50 MOI of Ad-NP was added to immature DCs (WT and 4-1BBL-/-
littermate controls, and 4-1BB-/-
) in 15 ml round bottom polypropylene tubes. Tubes were
centrifuged at 2200 rpm at 37°C for 2 hours. The next day, 1 μg/ml LPS was added to the
cultures. 24 hours following LPS treatment, DCs were washed three times with PBS, and
resuspended at a concentration of 10 × 106 cells/ml in PBS. 6-week-old recipient mice (DKO)
were intravenously injected with 1.5 × 106
DCs/mouse. On day 7 post DC vaccination, mice
were euthanized and their spleens were harvested. Splenocytes were surface stained with a
viability dye, anti-CD8-PE (clone 53-6.7; eBioscience), and Db/NP366-74 tetramers [obtained from
the National Institute for Allergy and Infectious Diseases tetramer facility (Emory University,
Atlanta, GA, USA)].
For intracellular staining, splenocytes were restimulated with 1 μM NP366-74 peptide for 6 hours
with Golgi Stop (BD Biosciences) at 37°C. For detection of degranulation, 5 μg/ml of anti-
CD107a (clone 1D4B; BD Biosciences) was also added at the beginning of the restimulation
culture. Cells were then surface stained with a viability dye and anti-CD8-PE. Following surface
staining, cells were fixed and permeabilized in Cytofix/Cytoperm solution (BD Biosciences) and
stained intracellularly with anti-IFNγ-PE-Cy7 (clone XMG1.2; eBioscience) and anti-TNF-APC
(clone MP6-XT22; BD Biosciences). FMO controls, unstimulated (no peptide), and naive
54
(unvaccinated mice) samples were used as staining controls. Samples were then analyzed by
flow cytometry.
2.3.8 Flow cytometry
Samples were analyzed using a FACSCalibur with CellQuest software or FACSCanto II with
FACSDiva acquisition software (BD Biosciences), and FlowJo software (Tree Star, Inc.).
2.3.9 Lentiviral transductions and confocal microscopy
A lentiviral vector (pLJM1) expressing 4-1BBL-citrine fusion protein or the empty vector, along
with a packaging plasmid, was first transfected into HEK293T cells using FuGENE6 (Promega).
48 hours following transfection, viral supernatants from the HEK293T cultures were used to
transduce WT and 4-1BB-/-
DCs on day 7 of DC culture. DC cultures with added viral
supernatants were spun at 2200 rpm at 37°C for 2 hours. Virally transduced DC cultures were
further stimulated with 1 μg/ml LPS, washed and visualized by confocal microscopy 24 hours
later. Cells were visualized in cell culture plates using an inverted Zeiss LSM510 confocal
microscope.
2.3.10 Statistical Analysis
All statistical analyses were performed using Graphpad software (Prism). For comparison of two
groups, p values were obtained using the Student’s t test (unpaired, two tailed, 95% confidence
interval).
2.4 Results
2.4.1 Chacterization of DCs lacking 4-1BB and 4-1BBL
To investigate the expression and function of 4-1BB and its ligand on DCs, we generated DCs
from bone marrow cells by culturing them in the presence of GM-CSF for 7-10 days (505).
Immature DCs derived from either WT, 4-1BB-/-
, or 4-1BBL-/-
bone marrow cells were identified
by CD11c/MHC-II double staining (Fig. 2.1A), and were found to express similar levels of
MHC-I and the costimulatory molecules CD80, CD86, and CD40 (Fig. 2.1B, top panel).
55
56
Following stimulation with 1 μg/ml LPS for 24 hours, WT, 4-1BB-/-
, and 4-1BBL-/-
DCs
similarly upregulated the expression of these molecules (Fig. 2.1B, bottom panel), suggesting
that 4-1BB and 4-1BBL are dispensable for LPS-induced upregulation of key DC surface
markers involved in APC function.
2.4.2 4-1BBL is not readily detectable in the presence of 4-1BB on LPS-activated DCs
We next investigated the expression of 4-1BB and 4-1BBL on LPS-activated bone marrow-
derived DCs. 4-1BB and 4-1BBL were not readily detectable on immature DCs (Fig. 2.2A), but
were both upregulated with LPS stimulation (Fig. 2.2B). 4-1BB was upregulated on WT DCs,
and in the absence of 4-1BBL on 4-1BBL-/-
DCs. However, interestingly, 4-1BBL was only
detectable in the absence of its receptor on 4-1BB-/-
DCs, and not on WT DCs. Thus, LPS
stimulation of DCs upregulates both 4-1BB and 4-1BBL, however, 4-1BBL is only detectable in
the absence of 4-1BB, suggesting that 4-1BB either downregulates 4-1BBL or masks its
detection.
2.4.3 The surface expression of 4-1BBL can be regulated extrinsically
Since 4-1BB and 4-1BBL can be expressed on the same cell type, it is possible that 4-1BB on the
same cell or on an adjacent cell regulates the expression/detection of 4-1BBL. To address
potential extrinsic regulation, immature CD45.2 4-1BB-/-
DCs were mixed with CD45.1 WT DCs
and stimulated with LPS for 24 hours. In this scenario, 4-1BBL expression on 4-1BB-/-
DCs can
only be regulated cell-extrinsically by 4-1BB on WT DCs. We found that 4-1BBL was readily
detectable on mature 4-1BB-/-
DC cultures in isolation (Fig. 2.3A, left panel), however, in the
mixed WT/4-1BB-/-
DC cultures, 4-1BBL was no longer detectable on the 4-1BB-/-
DCs (Fig.
2.3A, right panel). These data demonstrate that expression of 4-1BBL can be regulated or
masked in trans by 4-1BB on another cell. To confirm extrinsic regulation, we also added a
soluble fusion protein of 4-1BB (4-1BB-alkaline phosphatase, 4-1BB-AP) (439) to the mature 4-
1BBL-expressing 4-1BB-/-
DC cultures for 24 hours. Following addition of 4-1BB-AP, but not
AP alone, to the mature 4-1BB-/-
DC cultures, 4-1BBL was no longer detectable (Fig. 2.3B),
thereby providing further evidence that 4-1BB downregulates 4-1BBL or masks its detection.
57
58
59
2.4.4 Evidence of constitutive interaction between 4-1BB and 4-1BBL on DCs
In order to investigate the trafficking of 4-1BBL on DCs, we generated fluorescent fusion
proteins of 4-1BBL that were transduced into cell lines and DCs. Microscopy studies identified
mainly surface expression of 4-1BBL fusion proteins, even in the presence of 4-1BB (Fig. 2.4),
prompting us to further investigate whether 4-1BBL surface detection was masked in the
presence of 4-1BB. To test this, we conducted blocking experiments in both cell lines and DCs.
293T cells overexpressing 4-1BBL were pre-treated with either 4-1BB-AP, anti-4-1BBL (clone
TKS-1, used in figures 2.2-2.3), anti-4-1BBL (clone 19H3), Rat IgG, or PBS for 10 minutes on
ice to prevent potential signal-induced downregulation of 4-1BBL. The cells were then
separately stained with the 19H3 and TKS-1 anti-4-1BBL antibodies. When cells had been pre-
treated with either PBS or Rat IgG, 4-1BBL was similarly detectable by the two antibodies (Fig.
2.5A). However, in the presence of 4-1BB-AP (which binds to 4-1BBL), the level of 4-1BBL
detected by TKS-1 was significantly reduced, whereas 19H3 binding to 4-1BBL was
independent of the presence of receptor (Fig. 2.5A). These findings demonstrate that the TKS-1
binding site is masked by receptor binding, arguing that they bind at similar or overlapping sites.
On the other hand, the 19H3 epitope on 4-1BBL is at a distinct site from the binding site of 4-
1BB or TKS-1. Consistently, pre-treatment with 19H3 affected 19H3 binding, but not TKS-1
binding. However, pre-treatment with TKS-1 slightly affected 19H3 binding (Fig. 2.5A). Similar
results were obtained when LPS-treated 4-1BBL-expressing 4-1BB-/-
DCs were used. Once
again, in the presence of 4-1BB-AP, ligand was detectable by 19H3 but not by TKS-1 (Fig.
2.5B). The finding that the presence of 4-1BB prevents TKS-1 binding, but still allows 19H3
binding, argues that 4-1BBL is present on LPS-activated DCs in the presence of 4-1BB, but its
binding site for TKS-1 is masked. This leads to the conclusion that 4-1BB and 4-1BBL are
constitutively interacting on the DCs.
To further test this hypothesis, we examined 4-1BBL expression on LPS-activated WT, 4-1BB-/-
,
4-1BBL-/-
, and DKO DCs using both TKS-1 and 19H3 antibodies. 4-1BBL expression was
detected by 19H3 but not by TKS-1 on LPS-activated WT DCs (Fig. 2.5C), consistent with the
hypothesis that 4-1BBL is expressed on the surface of LPS-activated WT DCs, but undetectable
by TKS-1, because it is bound to 4-1BB. Taken together, these data provide evidence of
constitutive interaction between 4-1BB and 4-1BBL on LPS-activated DCs in vitro.
60
61
62
63
2.4.5 Indistinguishable in vitro survival of WT and DKO DCs
Given the finding of constitutive interaction between 4-1BB and 4-1BBL on LPS-activated DCs,
we next investigated the biological relevance of this interaction. 4-1BB plays a key survival role
on T cells (432, 433, 500) and there is also evidence that it plays a similar role on DCs (460,
464). To test the effect of constitutive 4-1BB/4-1BBL on the survival of LPS-activated DCs in
vitro, we monitored cell viability by live/dead stain, as well as by intracellular expression of the
pro-survival molecules Bcl-xL and Bcl-2 over a period of 5 days. There was no statistically
significant difference in the survival rate of WT and DKO DCs (Fig. 2.6A), arguing against a
key role for 4-1BB/4-1BBL in the in vitro survival of DCs following LPS stimulation. There was
a trend toward lower MFI of Bcl-xL in the DKO compared to WT DCs at each time point (Fig.
2.6B), as well as for Bcl-2 between 48 and 72 hours (Fig. 2.6C). However, these changes in MFI
did not affect overall DC survival, as WT and DKO DCs died at a similar rate.
2.4.6 4-1BB and/or 4-1BBL do not impact the level of the cytokines IL-2, IL-6, IL-12, IL-18,
and TNF, nor the chemokines MCP-1 and RANTES in LPS-activated DCs
Following stimulation with foreign antigen, DCs produce cytokines and chemokines that
orchestrate the inflammatory response. To ask whether 4-1BB and 4-1BBL contribute to pro-
inflammatory cytokine/chemokine production by DCs, we measured LPS-induced production of
five pro-inflammatory cytokines and two chemokines in WT and DKO/4-1BBL-/-
DCs. The
production of IL-2, IL-6, TNF, MCP-1, and RANTES were similarly upregulated with LPS
stimulation between WT and KO DCs (Fig. 2.7A, B). There was more variability in the
production of IL-12 and IL-18 between WT and DKO DCs, but no significant differences at any
of the time points assessed (Fig. 2.7A). Overall, these data demonstrate that 4-1BB and/or 4-
1BBL are dispensable for IL-2, IL-6, IL-12, IL-18, MCP-1, RANTES and TNF production by
LPS-activated DCs.
2.4.7 4-1BB and 4-1BBL are dispensable for antigen presentation by DCs in vitro
Arguably, one of the defining features of DCs is their ability to present antigen and stimulate
naive T cells, thus we sought to determine whether this key function of DCs is modulated by 4-
1BB/4-1BBL interaction. To ensure a high efficiency of antigen delivery, we delivered antigen
64
65
66
to the DCs using replication defective recombinant adenoviruses. To determine if 4-1BB/4-
1BBL could affect antigen uptake, WT and DKO DCs were transduced with different doses of
Ad-SIINFEKL-GFP and stimulated with LPS for 24 and 48 hours. The level of GFP expression
between the two groups was similar (Fig. 2.8A), suggesting that any observed differences in
antigen presentation were not due to differences in adenovirus-mediated antigen delivery. In
order to assay the capacity of the DCs to both process and present antigen, we next transduced
WT and DKO DCs with different doses of Ad-OVA for 24 and 48 hours in the presence of LPS,
and then looked at the expression of SIINFEKL peptide (OVA257-264) bound to MHC-I using an
MHC-restricted antibody. We did not find a statistically significant difference in the percentage
or MFI of SIINFEKL-MHC-I molecules as detected by anti-H-2Kb-SIINFEKL staining (Fig.
2.8B), suggesting that 4-1BB and 4-1BBL are dispensable for antigen processing by the DCs.
Next, we tested the ability of these cells to stimulate T cell responses. LPS-activated Ad-OVA
transduced DCs or SIINFEKL peptide-loaded DCs were mixed with CFSE-labelled OT-I CD8 T
cells at a 1:10 ratio of DCs to T cells. We tested two doses of both Ad-OVA and SIINFEKL
peptide at two time points (days 3 and 4), and found no striking difference in the CFSE dilution
profile of the T cells (Fig. 2.8C). We conducted similar experiments with OT-II cells to look at
CD4 T cell proliferation induced by DCs, and found no statistically significant difference
between WT and DKO DCs (data not shown). These findings demonstrate that the ability of DCs
to stimulate T cell proliferation in vitro is unaffected by the absence of 4-1BB/4-1BBL
interactions.
2.4.8 DC-intrinsic 4-1BB and 4-1BBL are largely dispensable for T cell priming against a
viral antigen in vivo
Although 4-1BB and 4-1BBL were dispensable on DCs for their in vitro activation of TCR
transgenic cells, it was conceivable that 4-1BB/4-1BBL had other effects in vivo and/or that
endogenous T cell responses would be more sensitive to the effects of 4-1BB/4-1BBL on DCs.
Therefore, we next investigated whether 4-1BB and 4-1BBL modulated DC immunogenicity in
vivo. To test this, in vitro generated WT, 4-1BB-/-
, and 4-1BBL-/-
DCs generated from bone
marrow of 4-1BB-/-
, and WT and 4-1BBL-/-
littermates, were modified with influenza NP (Ad-
NP) and used to prime mice (Fig. 2.9A). Of note, we titrated the dose of DCs, such that the
67
68
69
70
71
induced response to WT DCs was around 0.5% NP-specific CD8 T cells by day 7. As the natural
influenza infection produces a more than 10x higher response, this means that we are in a range
of response that is submaximal and should be sensitive to changes in the quality of the DCs. CD8
T cell responses were monitored, based on the previous evidence that the 4-1BB/4-1BBL
costimulatory pathway modulates the CD8 T cell response following influenza infection (436,
443). Moreover, previous studies have shown that addition of 4-1BB/4-1BBL signals during
priming enhances the NP-specific CD8 T cell response to influenza virus (502, 506, 507). In
order to deduce the DC-intrinsic effect of 4-1BB/4-1BBL, we used DKO hosts. At 7 days post
DC vaccination, we found no statistically significant difference in the proportions or numbers of
NP-tetramer-specific CD8 T cells (Fig. 2.9B), and we observed similar production of IFNγ (Fig.
2.9C) and TNF (data not shown), and expression of CD107a (data not shown) following re-
stimulation with NP peptide ex vivo. Taken together, these data show that 4-1BB and 4-1BBL
interact constitutively on DCs, but this interaction is dispensable for primary clonal expansion of
influenza-specific CD8 T cells.
72
2.5 Discussion
The activity of 4-1BB and 4-1BBL on DCs is poorly understood. Here we show that LPS-
activated DCs express both 4-1BB and 4-1BBL, and that these molecules constitutively interact
on this cell type. However, this interaction is dispensable for upregulation of key surface
molecules involved in APC function (MHC-I, CD80, CD86, CD40), survival,
cytokine/chemokine production (IL-2, IL-6, IL-12, IL-18, TNF, MCP-1, RANTES), in vitro
antigen processing and presentation to T cells, as well as in vivo priming against a viral antigen.
In the present study 4-1BBL was not readily detectable on LPS-activated WT DCs when using
the antibody TKS-1. 4-1BBL, however, was detected at high levels on LPS-activated 4-1BB-/-
DCs, suggesting that 4-1BB regulates the expression or detection of surface 4-1BBL. 4-1BBL
can be cleaved from the cell surface of leukocytes, and this cleavage is blocked by
metalloproteinase inhibitors in vitro (508). Enhanced levels of soluble 4-1BBL are found in the
sera of patients with leukemia and multiple sclerosis when compared to healthy controls (508,
509). Thus it was conceivable that 4-1BB could regulate 4-1BBL cleavage or could lead to
receptor-induced downregulation. Indeed, CD27 binding to CD70 results in reverse signaling
leading to downregulation of CD70 protein and mRNA levels (510). However, in the present
study, the major effect of 4-1BB on 4-1BBL was to block access of the antibody TKS-1. 4-1BBL
was still detected by the 19H3 antibody, arguing that 4-1BBL is present on the cell surface, but
that the TKS-1 epitope on 4-1BBL is masked by binding of its receptor.
The finding that 4-1BB and 4-1BBL interact constitutively on DCs does not distinguish whether
this interaction is between receptor and ligand on the same cells (in cis) or neighbouring cells (in
trans). 4-1BBL could be detected on 4-1BB-/-
cells but not WT cells within 3 hours following
LPS stimulation (data not shown) - suggesting a rapid interaction. Thus it is possible that 4-
1BB/4-1BBL interact within the same cell prior to surface exposure. Several studies have shown
that 4-1BB on T cells receives costimulatory signals (414), although the cell types that provide 4-
1BBL for these interactions have not been clearly delineated. If 4-1BB/4-1BBL interactions are
in trans, as was shown to occur in mixed DC cultures in the present study, then it is likely that
during DC-T cell interaction, that 4-1BBL on DCs could interact predominantly with 4-1BB on
T cells. However, if the interaction were cell-intrinsic, this might function to limit the 4-1BBL
signal available to the T cells. A previous study showed that 4-1BB and 4-1BBL transduced into
73
Jurkat T cells can interact in cis to activate NF-κB in a single cell assay (511). Using coculture
systems, the same study also showed that these molecules can interact in trans (511). Thus, 4-
1BB/4-1BBL activity on DCs could be mediated both within the same cell and between cells.
A key question raised by the finding of constitutive 4-1BB/4-1BBL interaction on DCs is how
this interaction modulates DC function. A previous study that used 4-1BB-/-
DCs stimulated with
LPS reported a pro-survival function of 4-1BB on DCs. Choi et al. showed that in the absence of
4-1BB, DCs had lower expression of Bcl-2 and Bcl-xL and a lower survival rate in vitro.
However, the differences in the survival rate between WT and 4-1BB-/-
DCs were quite marginal
(half-life of 36 vs. 24 hours), and only significantly increased with agonist anti-4-1BB
stimulation (460). In contrast, we find no striking effects of deficiency of 4-1BB or 4-1BBL on
the survival rate of DCs in vitro. Supraphysiological stimulation of 4-1BB on DCs, either by
anti-4-1BB antibody or in the presence of 4-1BBL overexpressing cell lines, has also shown a
role for this receptor in modulating CD80/CD86 expression, production of the cytokines IL-6,
IL-12 and TNF, and the ability to enhance in vitro T cell proliferation, independent of effects of
T cells (441, 462, 463). We show in this report that in contrast to their activity in response to
supraphysiological stimulation, endogenous 4-1BB and 4-1BBL do not modulate the ability of
DCs to mediate these functions, at least in the context of LPS-activated DCs. It is unlikely that
the concentrations of LPS used to stimulate DCs in the present study (0.01-1 μg/ml) were
suboptimal to see an effect of 4-1BB/4-1BBL on the DCs as previous studies that found an effect
of 4-1BB on DC survival and cytokine production used higher concentrations of LPS to
stimulate DCs [1-10μg/ml (462) and 5μg/ml (460)].
As antigen presentation and the ability to stimulate naive T cell expansion are hallmarks of DC
function, we further investigated whether DC-expressed 4-1BB and 4-1BBL contribute to this
function by using an in vivo model. We designed a DC vaccination experiment, in which
influenza NP was expressed in WT or 4-1BB/4-1BBL-deficient DCs, which were then used to
vaccinate mice lacking both 4-1BB and 4-1BBL. DKO mice were chosen as recipients in order
to assess the effects of 4-1BB/4-1BBL interaction only on the transferred DCs. We selected a
dose of antigen and DCs that induced an NP-specific CD8 T cell population that constituted
about 0.5% of CD8 T cells by day 7 following DC vaccination. This immunization dose was
chosen deliberately to be suboptimal such that it would be sensitive to effects of 4-1BB/4-1BBL
74
on outcome. However, under these conditions we found no significant role for 4-1BB/4-1BBL
interaction on the DCs in modulating the clonal expansion of influenza-specific CD8 T cells.
The influenza model was of interest as we have previously shown that incorporation of 4-1BBL
into an adenovirus vector containing influenza NP greatly augments the CD8 T cell response to
influenza (502). Bone marrow chimeras were used to show that these effects were largely due to
effects of 4-1BBL on T cells, although there was a small effect of 4-1BBL on non-T cells in this
model (502). The finding that we do not see an effect of 4-1BBL in the DC vaccination model
may again be due to use of endogenous 4-1BBL in the present study. Another caveat to this
vaccination model is that DCs used for vaccination may die rapidly and the contents cross-
presented via secondary DCs, in which case, 4-1BBL may not be relevant on the immunizing
DCs.
It should be noted that there is evidence in the literature that 4-1BB/4-1BBL interaction on
developing myeloid progenitor cells limits steady state myelopoiesis and differentiation into
CD11c+ DC lineages (470). The present study compared the numbers of CD11c-expressing cells
generated from WT and 4-1BBL-/-
littermate bone marrow derived DC cultures (data not shown),
but did not identify a role for these molecules in limiting DC differentiation. It is possible that
our DC culture conditions were not sensitive enough to observe these differences, as we
developed progenitor cells in the presence of 40 ng/ml of GM-CSF, whereas the aforementioned
study used 10 ng/ml of GM-CSF (470). However, it is not clear in (470) whether appropriate
littermate controls were used to rule out effects of incomplete backcrossing and the microflora,
which we did control for in the present study. Anti-4-1BB stimulation of DCs has also been
suggested to suppress T cell immunity. In an oral tolerance model, anti-4-1BB stimulation of
GALT DCs was suggested to promote the development of iTreg (461). Furthermore, early
administration of anti-4-1BB in the context of LCMV Armstrong infection was shown to target
both DCs and T cells, and result in AICD of CD8 T cells (463). It is possible that the 4-1BB
pathway in DCs augments the ability of DCs to induce Tregs or mediate T cell apoptosis;
however, we did not see an effect of endogenous DC-intrinsic 4-1BB in modulating CD8 T cell
responses in a very sensitive in vivo model. These studies (461, 463) investigated
supraphysiological stimulation of 4-1BB on DCs, which we have shown here does not
necessarily compare with the endogenous function of this molecule.
75
Overall, this study provides evidence of constitutive interaction between 4-1BB and 4-1BBL on
LPS-activated DCs. Although we have not identified a physiological role for this interaction, the
finding that this interaction is so complete in DC cultures as to fully mask 4-1BBL when probed
with antibodies that target the receptor binding site, makes it hard to believe it does not play a
role in DC biology. These data highlight the need for further investigation into the role of
endogenous 4-1BB/4-1BBL expression and interaction on DC biology. As the 4-1BB/4-1BBL
pathway is a target in various anti-tumor and anti-viral therapeutic strategies, it is important that
we understand how these molecules impact not only T cell biology, but the biology of other cell
types that express these molecules, including DCs.
76
Chapter 3
Natural killer cells contribute to splenic atrophy observed months following
LCMV clone 13 infection
Mbanwi AN, Wang C, Geddes K, Philpott DJ, Watts TH. Natural killer cells contribute to
splenic atrophy observed months following LCMV clone 13 infection.
Manuscript in preparation.
Author Contributions
Mbanwi AN and Watts TH designed the experiments and wrote the paper.
Wang C made the observation that spleens are small following LCMV clone 13 infection and
contributed data points to Fig 3.1A and E.
Geddes K prepared overnight cultures of Salmonella and infected mice with Salmonella.
Philpott DJ provided Salmonella and Salmonella-infected mice were maintained under Dr.
Philpott’s permit.
Mbanwi AN conducted all other experiments.
77
3.1 Abstract
Lymphocytic choriomeningitis virus (LCMV) clone 13 infection of mice is a widely used model
for investigating the mechanisms driving persistent viral infection in humans. Early during
LCMV clone 13 infection, there is a disruption of splenic architecture, which then returns to
normal within a few weeks. However, the long-term effects of clone 13 infection on splenic
structure have not been reported. Here, we report that persistent infection with LCMV clone 13
results in sustained splenic atrophy that persists for up to 10 months following infection, whereas
infection with the acutely infecting LCMV Armstrong is associated with a return to pre-infection
spleen weights. The pathophysiological mechanisms underlying splenic atrophy often found in
autoimmune conditions such as celiac disease are not known. By blocking NK cells at the onset
of splenic atrophy (19-21 dpi), we show that NK cells contribute to the dramatic late-stage loss
in spleen size and cell number. Compared to LCMV clone 13-infected isotype control antibody
treated mice, treatment of LCMV clone 13-infected mice with a type I interferon receptor 1
(IFNAR1) blocking antibody at the onset of splenic atrophy significantly increased the
proportion of NK cells in the spleen and exacerbated splenic contraction around 2 months
following initial infection. LCMV clone 13 infection and associated atrophy of the spleen were
shown to compromise subsequent immunity to a bacterial pathogen. These findings implicate
NK cells in the pathophysiological mechanisms underlying splenic atrophy following viral
infection, raising the possibility that NK cells contribute to splenic atrophy in other diseases.
3.2 Introduction
LCMV is a non-cytopathic rodent pathogen. Infection with LCMV Armstrong leads to a robust
CD8 T cell response that results in rapid clearance of this acutely infecting strain of the virus.
LCMV clone 13 is a variant of LCMV Armstrong, isolated by serial passage in mice (258), that
differs by only 3 amino acids (267), resulting in a persistent viral infection, that is ultimately
cleared from most organs by 60-90 dpi. LCMV clone 13 infection of mice is a widely used
model to study the mechanisms driving persistent viral infection and has provided valuable
insights into how such mechanisms could be manipulated to treat chronic human viral disease
and other chronic conditions, such as cancer (437, 512-517). Despite the lack of cell lysis
78
induced by the virus itself, chronic infection with LCMV is associated with specific and
generalized immunosuppression (518) due to induction of immunoregulatory mechanisms as a
consequence of persistent immune stimulation. This immune regulation leads to functional
exhaustion of the immune system (295, 519). Included in these outcomes is the loss of function
as well as early depletion of anti-viral T cells, culminating in poor viral control.
Previous studies have shown that infection with various strains of LCMV, including WE and
clone 13, leads to disruption of lymphoid architecture, a process that is reversed after viral
clearance (363, 366). However, the long term effect of LCMV clone 13 on splenic structure has
not been reported. Here we note a remarkable difference in the size of the spleen and the number
of immune cells following clone 13 as compared to Armstrong infection of mice. In contrast to
Armstrong infection, which induces transient splenomegaly, with a return to baseline spleen size
after viral clearance, clone 13-infected mice exhibit extensive and sustained splenic atrophy and
lymphopenia persisting up to 10 months following initial infection, well after virus can no longer
be detected in the spleen, kidney or liver. Here, we show that the reduced spleen size following
chronic viral infection is also associated with delayed control of a subsequent bacterial infection.
Splenic atrophy and the resulting perturbation in anti-bacterial immunity is a major issue in
autoimmune conditions such as celiac disease (229). The mechanisms causing splenic atrophy in
autoimmunity or following infection are ill-defined.
NK cells are cytotoxic lymphocytes that recognize their targets via an array of both activating
and inhibitory receptors, and mediate their effector functions by release of perforin and
granzymes and cytokines such as IFNγ and TNF (91, 520). In the context of LCMV infection,
NK cells were recently shown to play a regulatory role in the first few days following infection,
as NK cell depletion prior to infection increased anti-viral T cell responses and improved viral
control (322-324). However, the role of NK cells late in the infection is not clear. Here, we
implicate NK cells in modulating late-stage splenic atrophy following LCMV clone 13 infection.
NK cell depletion at 3 weeks pi, the time when splenic atrophy is first noted, significantly
ameliorated splenic contraction and loss of cell numbers. IFNAR1 blocking antibody treatment
at the onset of splenic atrophy dramatically increased the proportion of NK cells compared to
isotype control antibody treatment, which correlated with exacerbated splenic contraction and
79
decreased cell recovery from the spleen. These findings implicate NK cells as contributing to
splenic atrophy following viral infection.
3.3 Materials and Methods
3.3.1 Mice and infections
5-6 week old C57BL/6 (B6) mice purchased from Charles River Laboratories (Saint-Constant,
QC, Canada) were infected intravenously with either 2 × 106 PFU of LCMV clone 13 or 5 × 10
3
PFU of LCMV Armstrong. Armstrong and clone 13 strains were prepared and quantitated as
previously described (258). Naive mice were either assessed on day 0 at 5-6 weeks old, or aged
to correspond with late-stage LCMV-infected mice. Mice were euthanized, and spleen weights
and number of splenocytes (as determined by trypan blue exclusion counting) were tracked from
days 0-300 for LCMV clone 13 infections, and from days 0-80 for LCMV Armstrong infections.
LCMV titers in the spleen, liver, and kidney were determined by a plaque-forming assay as
previously described (521).
Overnight cultures of streptomycin-resistant Salmonella enterica serovar Typhimurium (SL1344
ΔaroA) were washed two times with PBS, and the concentration of bacteria was measured by
determining the optical density at 600 nm. 14-15 week old naive B6 mice and B6 mice that had
been infected with 2 × 106 PFU of LCMV clone 13 2 months prior were infected
intraperitoneally with 105 CFU of Salmonella. 7-14 days post Salmonella infection, mice were
euthanized, and spleens and livers were homogenized in PBS containing 1% Triton X-100 with a
rotor homogenizer. Spleen and liver homogenates were serially diluted in PBS and plated on
Luria-Bertani agar plates containing 50 μg/ml streptomycin. Colonies were counted after
overnight incubation at 37°C.
All mice were maintained under specific pathogen-free conditions in sterile microisolators at the
University of Toronto. All animal studies were approved by the University of Toronto animal
care committee in accordance with the regulations of the Canadian Council on Animal Care.
3.3.2 In vivo antibody treatments
80
On days 19 and 21 post LCMV clone 13 infection, B6 mice were injected intravenously with 0.2
mg/mouse of Rat IgG control (Sigma-Aldrich) or anti-NK1.1 [clone PK136, hybridoma kindly
provided by Dr. Pamela Ohashi (Ontario Cancer Institute, University Health Network, Toronto,
ON, Canada)] in PBS.
On days 19 and 21 post LCMV clone 13 infection, B6 mice were injected intraperitoneally with
500 μg of a mouse IgG1 isotype control (anti-TNP; clone 1B711) or IFNAR1 blocking antibody
(clone MAR1-5A3; Bio X Cell, West Lebanon, NH, USA). On day 23 post clone 13 infection,
mice received a final intraperitoneal injection of 250 μg of control or IFNAR1 blocking
antibody.
3.3.3 Flow cytometry and antibodies
Single cell suspensions of splenocytes were surface stained with antibodies, including anti-
TCRβ-PE-Cy5 (clone H57-597), B220-eFluor 450 (clone RA3-6B2), NK1.1-FITC (clone
PK136), NKp46-PE (clone 29A1.4), biotinylated NKG2D (clone A10) and streptavidin-PE,
MULT1-PE (clone 5D10), CD4-PE-Cy5 (clone RM4-5; BD Biosciences), and CD3-PE-Cy7
(clone 145-2C11). Intracellular staining was performed using Cytofix/Cytoperm solution (BD
Biosciences) and anti-RORγt-PE (clone B2D). All antibodies were purchased from eBioscience,
unless otherwise noted. Cell viability was determined using a LIVE/DEAD Violet
Viability/Vitality Kit (Life Technologies). Fluorescence minus one controls were used as
negative controls for antibody staining. Samples were analyzed using FACSCanto II (BD
Biosciences) with FACSDiva acquisition software. Data analysis was performed using FlowJo
software (Tree Star, Inc.).
For detection of LTi, spleens were cut into small pieces and treated with collagenase IV for 45
minutes at 37°C in a bacterial shaker. Spleen samples were then pipetted up and down to create a
single cell suspension. Splenocytes were washed with PBS, counted, and stained with the
appropriate antibodies.
3.3.4 Immunofluorescence microscopy
Spleens from naive and LCMV clone 13-infected B6 mice were removed from the animals and
frozen in Optimal Cutting Temperature (OCT) compound (Sakura Finetek). Spleen tissue
81
sections were then cut at 6-8 microns using a Leica CM3050 cryostat (Leica Microsystems),
mounted on glass microscope slides and fixed in acetone. Sections were blocked with TBS-T
[TBS containing 0.05% Tween-20 (Sigma-Aldrich)] supplemented with 10% normal mouse
serum (The Jackson Laboratory) and 2 μg ml-1
anti-CD16/CD32 Fc block (clone 93;
eBioscience) for 30 minutes. Sections were stained using the following antibodies: anti-Thy1.2-
PE (clone 30-H12), B220-FITC (clone RA3-6B2), CD31-APC (clone 390), VCAM-1-Alexa
Fluor 647 (clone 429), biotinylated CD21/CD35 (clone 8D9) followed by streptavidin-PE in a
second step, and isotype control antibodies. Stains were performed in the dark for at least 1 hour.
All antibodies were purchased from eBioscience, unless otherwise noted. Slides were washed 3
times with TBS-T, followed by a final wash in PBS. DAPI nucleic acid stain (Invitrogen) was
then applied to the slides for 30 seconds, and washed off 3 times with PBS. Slides were then
mounted with Gel/Mount (Biomeda Corp.) and images were acquired with a Leica DMRA2
microscope (Leica Microsystems) equipped with a Retiga EXi digital camera (Q Imaging) using
OpenLab software (Improvision).
3.3.5 Statistical analysis
All statistical analyses were performed using Graphpad software (Prism). For comparison of two
groups, p values were obtained using the Student’s t test (unpaired, two tailed, 95% confidence
interval). Statistically significant differences are indicated as *, p < 0.05, **, p < 0.01 and ***, p
< 0.001.
3.4 Results
3.4.1 Persistent splenic atrophy following infection with LCMV clone 13
In the course of studying the resolution phase of the immune response in mice infected with
LCMV clone 13, we noted dramatic splenic atrophy at 60 days following infection, when
compared to pre-infection spleen (Fig. 3.1A left). Atrophic spleens at this time point were 64%
of their pre-infection weight (median of 93.7 mg on day 0 compared to a median of 59.8 on days
60-70 following infection, p < 0.0001). To ascertain the kinetics of this atrophy, spleen weight
and number of splenocytes were tracked from days 0-300 following infection with LCMV clone
82
83
13 (Fig. 3.1A center and right). Following infection with LCMV clone 13, spleen size increased
to a median weight of 203.7 mg by day 8. After reaching a peak size at day 8, spleen weight
decreased between days 8 and 21 to just below baseline and continued to decrease between days
35 and 50, thereafter maintaining a median weight of around 60 mg, and remained significantly
below baseline weight out to day 300 (Fig. 3.1A center). In addition, at 300 dpi, inguinal LNs in
LCMV-infected mice were about half the size of the inguinal LNs in naive animals (data not
shown). The size of the spleen was mirrored by the total number of splenocytes recovered at
each time point (Fig. 3.1A right). Over this time period, the overall weight of the mice increased
as expected, therefore splenic atrophy was not due to an overall cachexia in the animal (Fig.
3.1B). These data show that mice undergo splenic atrophy at days 21-50 pi and that the spleens
fail to recover to normal pre-infection weights up to 10 months pi.
To rule out the possibility that aging of the infected mice, compared to their 5-6 week naive
counterparts was responsible for splenic atrophy, we repeated this analysis with age-matched
uninfected controls (Fig. 3.1C). Naive WT age-matched mice had slightly bigger spleens than
their naive 5-6 week old counterparts (medians of 106.2 compared to 93.7 mg, p = 0.0809),
indicating that splenic atrophy is not a normal process associated with aging, rather that healthy
aging normally results in an increase in spleen size, and that splenic atrophy is a result of the
persistent infection.
We next asked whether splenic atrophy also occurred with an acutely infecting LCMV variant,
LCMV Armstrong, or whether it was a feature of more prolonged infection. We found a similar
increase in spleen weight by 8 dpi between clone 13 and Armstrong, however with Armstrong
infection, spleens contracted back to pre-infection weight around 30 dpi, and maintained this
weight up to 80 dpi (Fig. 3.1D). These findings suggest that persistent, but not acute infection,
results in late-stage splenic atrophy.
It was possible that splenic atrophy observed following LCMV clone 13 infection was due to the
persistence of the virus at late time points. To address this, we assessed viral load at time points
when sustained splenic atrophy is observed using plaque assays on the spleen, kidney and liver
of infected mice. Virus was detectable at 8 dpi in the spleen, but largely cleared by day 60, and
undetectable in the spleen thereafter (Fig. 3.1E), consistent with previous reports. At late time
points (80 dpi +), virus was no longer detectable in the kidney and liver (data not shown). These
84
data suggest that the continued presence of virus is not required to sustain splenic atrophy, as the
spleen size fails to recover after virus is no longer detectable in spleen, liver or kidney.
3.4.2 Atrophic spleens are lymphopenic, but show T cell/B cell segregation within the white
pulp
To assess the impact of splenic atrophy on the cellular composition of the spleen, we compared
the proportion and number of immune cells in age-matched naive and 60 days post LCMV-
infected mice, as well as in naive 5-6 week old mice (Fig. 3.2A). We observed a significant
increase in the proportion of T cells in the late-stage LCMV-infected mice compared to young
and age-matched groups of naive mice, and a concomitant decrease in the proportion of B cells.
The proportion of non-B non-T cells was not significantly different between naive and infected
(Fig. 3.2A). However, when converted to total numbers, it was clear that, there were significantly
less T, B, and non-B non-T cells in the atrophic spleens. We further characterized the non-B non-
T cell population with antibodies to CD11b, CD11c, MHC-II (antigen presenting cells), F4/80
(macrophages), NK1.1 (NK cells), and Gr-1 (granulocytes), and similarly found a significant
reduction in the number of these cells in atrophic spleens (data not shown). Overall, these
findings show that splenic atrophy is due to an overall decrease in the numbers of T, B and non-
B non-T cells in the spleen. However, based on frequency, the B cell compartment seems to be
disproportionately affected compared to the T cell compartment.
Persistent LCMV infection has been shown to disrupt splenic architecture early following
infection, with recovery between days 14-20 pi (363). However, at these time points, maximal
splenic atrophy has not yet occurred. Thus, we investigated the organization of the spleen at late
time points when there is maximal and sustained splenic atrophy. Thy1.2, B220, and CD31 were
used to define T cells, B cells, and endothelial cells, respectively. CD35, a marker of follicular
dendritic cells (FDCs) and B cells, and VCAM-1 were used to look at stromal cells in the spleen.
We found T cell/B cell segregation, and similar stromal cell networks between day 60 LCMV-
infected and naive mice (Fig. 3.2B). Thus splenic atrophy persists despite the appearance of
organization within the white pulp at late time points following clone 13 infection.
85
86
3.4.3 NK cells contribute to splenic atrophy following LCMV clone 13 infection
NK cells have been implicated in the resolution phase of the immune response (522), and have
been identified as critical players both early and late following LCMV clone 13 infection, where
they were suggested to eliminate virus-specific T cells and promote viral persistence (196, 322-
324). Thus, to evaluate whether NK cells were participating in the drastic loss of spleen cells, we
evaluated the effect of NK cell depletion on splenic atrophy following clone 13 infection. Mice
were infected with LCMV clone 13 on day 0, and then on days 19 and 21 pi, the time point when
spleen size returns to baseline just before undergoing further atrophy, we treated mice with either
an NK cell-depleting antibody (anti-NK1.1) or an isotype control antibody (Fig. 3.3A). The
extent of NK cell depletion was measured in the blood at 25 dpi (69% depletion), and in the
spleen on days 35 (80% depletion) and 50 (47% depletion) following infection (Fig. 3.3B). NK
cells were largely depleted up to day 35 following infection, but their numbers started to recover
by day 50. Spleen weight and cell numbers were assessed on day 50 following infection, which
corresponded to around 1 month following NK cell depletion (Fig. 3.3C). On day 50 pi, mice
that had been treated with NK cell-depleting antibody on days 19 and 21 pi showed a 22%
increase in spleen weight compared to mice treated with control antibody (median of 62.3 mg for
control mice, and median of 75.7 mg for NK cell-depleted mice, p = 0.0001), as well as a 70%
increase in the number of cells in the spleen (median of 54 × 106 cells in control mice and 92 ×
106 cells in NK cell-depleted mice, p < 0.0001) (Fig. 3.3C). Compared to the median spleen
weight on day 21 pi (81.9 mg), control treated mice experienced a 24% reduction in spleen
weight (median of 62.3 mg), whereas NK cell-depleted mice only experienced a 7.6% reduction
in spleen weight (75.7 mg). NK cell depletion did not change the proportion of cells in the
spleen, but did increase the number of T, B, and non-B non-T cells (data not shown). Thus, these
findings suggest that NK cells contribute to splenic atrophy following persistent LCMV
infection.
We next asked how NK cells could be contributing to splenic atrophy. NKG2D is an activating
receptor on NK cells that was recently implicated in the regulatory functions of NK cells
following LCMV infection (322). As the expression of NKG2D ligands renders cells susceptible
to NK cell-mediated lysis, we looked at the expression of the NKG2D ligands, MULT1 and Rae-
1δ, 50 days-post LCMV clone 13 infection, however, we did not detect expression in the spleen
87
88
89
at this time point (data not shown). We speculated that by day 50, NKG2D ligand-expressing
cells would have been depleted, and thus looked on day 21, a time point at which the spleen has
started to contract below baseline, but has not yet reached the maximal contraction weight. On
day 21 pi, NK cells, as defined by NKp46 expression, expressed NKG2D (Fig. 3.3D), and
MULT1 expression was found predominantly on B cells (Fig. 3.3E). Rae-1δ expression was not
detectable (data not shown). These data point to a possible role for NKG2D-mediated lysis of
MULT1-expressing B cells, in splenic atrophy following LCMV clone 13 infection.
3.4.4 IFNAR1 blocking antibody treatment increases the proportion of NK cells, and
exacerbates splenic atrophy
Chronic infection results in high levels of type I IFNs. As NK cells can be activated by type I
IFNs (344), and chronic IFN signaling plays a regulatory role following LCMV clone 13
infection (320, 353), we asked whether IFN signaling contributes to the process of splenic
atrophy following LCMV clone 13 infection. To this end, mice were infected with LCMV clone
13 on day 0, and then treated with either IFNAR1 blocking (320, 353) or isotype control
antibody on days 19, 21, and 23 (Fig. 3.4A). Spleen weights, number of splenocytes, and cell
populations were assessed about a month later on day 50. Surprisingly, spleens from IFNAR1
blocking antibody treated mice had an 18% decrease in spleen weight compared to isotype
control antibody treated mice (medians of 68.5 mg and 56.5 mg in isotype control treated and
IFNAR1 blocking antibody treated mice, respectively, p < 0.0001), as well as a 44% decrease in
cell numbers (medians of 110 × 106 cells and 62 × 10
6 cells in isotype control antibody treated
and IFNAR1 blocking antibody treated mice, respectively, p = 0.0002) (Fig. 3.4B). Compared to
the median spleen weight on day 21 pi (81.9 mg), isotype control antibody treated mice
experienced a 16% decrease in spleen weight (median of 68.5), while IFNAR1 blocking
antibody treated mice experienced a 31% decrease in spleen weight (median of 56.5 mg). In
terms of proportion of cells on day 50 pi, there was a significant decrease in the proportion of B
cells, and a concomitant increase in the proportion of T cells in the IFNAR1 blocking antibody
treated mice compared to isotype control antibody treated mice (Fig. 3.4C), whereas there was
no statistically significant difference in the proportion of non-B non-T cells (Fig. 3.4C). When
converted to absolute numbers, there were significantly fewer T, B, and non-B non-T cells in the
IFNAR1 blocking antibody treated mice compared to isotype control antibody treated mice (Fig.
90
91
92
3.4C). To understand these findings, we looked at the proportion of NK cells, and found an 87%
increase in the proportion of NK cells in IFNAR1 blocking antibody treated compared to isotype
control antibody treated mice (Fig. 3.4D, 3.4E). The cells in the IFNAR1 blocking antibody
treated mice also had a higher MFI of NKG2D, suggesting increased functionality (Fig. 3.4E).
Thus, compared to isotype control antibody treatment, IFNAR1 blocking antibody treatment
during persistent LCMV infection resulted in increased proportions of NK cells, with evidence of
increased activation, correlating with increased splenic atrophy. These data further support a role
for NK cells in splenic contraction at the late stages of LCMV clone 13 infection.
As the type I IFN response did not appear to account for splenic atrophy following LCMV clone
13 infection, in fact the opposite was observed, we asked if other cytokines might contribute. As
TNF is a cytokine associated with cell death and cachexia, we also measured the spleen weights
and counted the number of cells in TNF-/-
mice at 60 days post LCMV clone 13 infection. Mice
lacking 4-1BBL were also included as 4-1BBL has been shown to regulate TNF levels in
macrophages (440). However, similar to WT mice, TNF-/-
and 4-1BBL-/-
mice had small spleens
(medians of 46.1 and 60.5 mg, respectively), suggesting that neither TNF, nor 4-1BBL,
contribute significantly to late-stage splenic atrophy (data not shown).
3.4.5 Splenic atrophy delays clearance of a bacterial pathogen
Splenectomy, hyposplenism, and asplenia have been shown to greatly increase susceptibility to
certain bacterial infections in humans (248). Therefore we asked whether splenic atrophy
following persistent viral infection compromises subsequent anti-bacterial immunity. To
investigate the response to secondary bacterial challenge, age-matched naive and 60 days-post
LCMV-infected mice were infected with a high dose of an avirulent aroA mutant strain of
Salmonella typhimurium. 7 days post Salmonella infection, the bacterial burden in the spleen and
liver were assessed. An attenuated strain of Salmonella was chosen as the virulent strain of
Salmonella killed the mice too quickly to assess bacterial burden with or without prior LCMV
infection. We found that mice that had been previously infected with LCMV clone 13 had about
a 3-fold increase in the bacterial load in the spleen (Fig. 3.5A) and liver (Fig. 3.5B), compared to
age-matched naive mice, although by day 14, both naive and LCMV clone 13-infected mice
largely cleared the attenuated pathogen. These findings demonstrate that infection with LCMV
93
94
clone 13 with its associated splenic atrophy can delay subsequent control of Salmonella
infection.
3.4.6 The proportion of LTi is similar between aged naive and LCMV-infected mice
It is remarkable that months following LCMV clone 13 infection and viral clearance from the
spleen, liver, and kidney, atrophied spleens do not recover to pre-infection weight. Lymphoid
tissue inducer cells (LTi) fall into the category of group 3 innate lymphoid cells (ILCs), which
are capable of producing IL-17A and/or IL-22, and depend on the transcription factor RORγt for
their development and function (90). LTi are critical in LN and Peyer’s patch organogenesis in
the embryo and post-natal period (523, 524). Adult LTi were recently shown to participate in the
restoration of lymphoid tissue integrity following LCMV WE infection (370). To ask whether
the lack of spleen recovery following LCMV clone 13 infection is due to a potential depletion of
LTi, we infected mice with clone 13, and assessed both the proportion and number of LTi
(CD4+CD3
-CD11c
-B220
-RORγt
+) in the spleen 300 days later (Fig. 3.6). LCMV-infected mice
were compared to naive mice aged between 3 and 9 months. Our findings show that the
proportion of LTi is similar between naive and LCMV-infected mice. When converted to total
number, there is a trend (p = 0.1567) of lower numbers in the LCMV-infected group, likely due
to the overall loss in the number of splenocytes. It should be noted that our analysis of LTi could
also include the recently identified subset of group 3 ILCs, NCR+ILC3s, which also express
RORγt and can express CD4, but unlike LTi, express NKp46 (90). Taken together, it appears
that late-stage LCMV infection does not affect the proportion of LTi or LTi-like cells present in
the spleen.
95
96
3.5 Discussion
The mechanisms governing the resolution of inflammation following LCMV infection are ill-
defined. Both LCMV Armstrong and LCMV clone 13 infections result in a substantial immune
response that initially doubles the size of the spleen. Following LCMV clone 13 infection, the
spleen contracts up to 50% of its pre-infection weight months following infection, whereas
spleens from LCMV Armstrong-infected mice return to their pre-infection weights. We observed
sustained splenic atrophy following LCMV clone 13 infection up to 10 months following
infection, a time point several months after virus is no longer detectable in the spleen, kidney and
liver. Atrophic spleens were lymphopenic, but did not show gross abnormalities in architecture.
Using an NK cell-depleting antibody at the onset of splenic atrophy, we provide evidence that
NK cells contribute to splenic atrophy. In keeping with the highest expression of the NKG2D
ligand, MULT1, on the B cells 21 dpi, B cells showed the biggest detriment in numbers over the
naive spleen. Thus it appears that NK cells contribute to splenic atrophy by impacting T cells, B
cells and non-B non-T cells, but B cells may be most susceptible. IFNAR1 blocking antibody
treatment at the onset of splenic atrophy increased the proportion of NK cells in the spleen,
which correlated with exacerbated splenic atrophy. Moreover, LCMV clone 13 infection and
associated splenic atrophy resulted in compromised immunity when mice were challenged with
an attenuated strain of Salmonella.
We found that mice undergo sustained splenic atrophy months following persistent, but not
acute, infection with LCMV. Persistent viral replication appears to initiate a program that results
in splenic atrophy at late stages following initial infection – a program that is distinct from that
induced by acute viral replication. It is well known that persistent as opposed to acute LCMV
infection, induces a state of generalized and specific immune suppression that is associated with
increased immune regulation, virus-specific T cell dysfunction/exhaustion and deletion, and
subsequent poor viral control (2). This poor control of virus following clone 13, but not
Armstrong infection, could lead to the excessive bystander activation of cells in the spleen,
thereby making these cells susceptible to either intrinsic or extrinsic forms of cell death. It is
unlikely that direct infection of T and B cells, followed by lysis of infected cells, mediated their
removal as these cells have very low expression of the cellular receptor for LCMV, α-
dystroglycan, and clone 13 has been shown to preferentially infect dendritic cells (257, 270).
97
The mechanisms underlying late-stage atrophy observed with LCMV clone 13 have not been
previously reported. The NK cell depletion studies reported here indicate that NK cells are either
directly or indirectly contributing to splenic atrophy late following LCMV clone 13 infection.
NK cells were recently implicated in the resolution phase of a self-limited asthma model in mice.
Either depleting NK cells or blocking their migration to target tissues delayed clearance of
eosinophils and antigen-specific CD4 T cells (522). In the context of LCMV clone 13 and/or WE
infection, several groups have identified an immunoregulatory role for NK cells both early (322-
324) and later (196) in the infection. Depleting NK cells prior to infection (322-324) and
delaying NK cell depletion from 1 day to 4 weeks pi (196) increased virus-specific T cell
responses and improved viral control. Waggoner et al. point to a direct effect of NK cells on
CD4 T cells (196, 323), which then impact the CD8 T cell response, whereas Lang et al. point to
a direct effect of NK cells on CD8 T cells (322). However, these studies (196, 322-324) do not
report on the effects of NK cell depletion on spleen size, or on the long term outcome of these
effects.
The receptor-ligand interactions underlying NK cell recognition and depletion of T cells
following LCMV clone 13 infection are not clear, but might involve the activating NK cell
receptor, NKG2D. In the context of LCMV WE infection, Lang et al. point to a role for NKG2D
in the recognition and early removal of virus-specific CD8 T cells (322). Ligands for the
activating receptor NCR1, and the inhibitory ligands MHC-I, Qa-1b, and CD48 have also been
implicated in recognition of T cells by NK cells (325, 327, 360). The present study identifies an
effect of NK cells on not only T cells, but on several other immune cell types during the
resolution phase of persistent LCMV infection. Notably, our finding that NK cells express
NKG2D and that B cells express the NKG2D ligand, MULT1, at the onset of splenic atrophy,
identifies B cells as a potential target for NK cell-mediated killing. These data likely explain why
late-stage LCMV clone 13-infected mice have a lower proportion of B cells in the spleen. On
day 21 pi, MULT1 expression was not readily detectable on T cells and non-B non-T cells. This
could have been due to these cell types having lower levels of the ligand on day 21 pi, acquiring
ligand expression and being depleted prior to day 21, or acquiring ligand expression after day 21
and being depleted at a later time point. It is also possible that the receptor-ligand interactions
98
that mediate B cell depletion are distinct from those that mediate T cell and non-B non-T cell
depletion.
Our finding that IFNAR1 blocking antibody treatment (compared to isotype control antibody
treatment) at the onset of splenic atrophy increases the proportion of NK cells in the spleen is
consistent with a role for type I IFNs in limiting the accumulation of NK cells at later stages of
LCMV clone 13 infection. Type I IFNs are known activators of NK cell effector function during
viral infection (344), but have been shown to exert anti-proliferative effects on mouse and human
NK cells in vitro (347, 525, 526). Although there is evidence that type I IFNs augment mouse
NK cell proliferation in vivo (347), this was later attributed to a role for these cytokines in
inducing IL-15 (527). It is possible that persistent IFN signaling induced by LCMV clone 13
infection (320, 353) coupled with potentially differential induction of IL-15 or other cytokines at
different stages of infection, normally limits the number of splenic NK cells present at later
stages of infection. Regardless of mechanism, the finding that IFNAR1 blocking antibody
treatment results in an increased proportion of NK cells and exacerbates splenic contraction at
late time points, adds further evidence for a role of NK cells in splenic atrophy.
The finding that spleens from LCMV clone 13-infected mice do not return to pre-infection
weights up to 10 months following infection raised the question of whether these mice have
compromised immunity. As humans with splenic atrophy or those that have undergone
splenectomy are more susceptible to certain bacterial infections (248), we tested the effect of
prior LCMV clone 13 infection and splenic atrophy on control of a bacterial infection. Previous
studies have shown that infection of mice with LCMV WE led to significantly higher bacterial
loads in the spleen and liver following challenge with either Listeria monocytogenes, S. aureus,
or Salmonella typhimurium when compared to LCMV-naive mice. However, in this study, mice
were challenged 2 days following LCMV infection (528), a time point when virus is still present.
Here, we investigated challenge during late stages of LCMV infection, at time points when virus
is largely cleared from the spleen and undetectable in the liver in our model, to look not at the
effect of viral replication per se, but at the effect of spleen size. We found that when mice were
challenged with Salmonella at 60 days following LCMV clone 13 infection, they had a higher
bacterial burden than Salmonella-challenged LCMV-naive mice, suggesting that LCMV clone
13-induced splenic atrophy compromises subsequent immunity.
99
A key question that our study raises is why the spleen fails to recover to pre-infection weight
after NK cells have removed the activated/stressed cells and virus has been cleared. Is it possible
that LCMV infection and subsequent cell loss leads to the depletion of a cell type required for
lymphoid tissue homeostasis? A previous study (362) has shown that a lymphotoxin (LT) and B
cell dependent pathway is responsible for peripheral LN expansion following LCMV WE
infection. Similarly, LT-expressing LTi were shown to participate in the restoration of lymphoid
organ structure following LCMV infection (370). These cells were of interest because of their
critical role during LN and spleen organogenesis in the embryo and post-natal period (523, 524).
However, as our data suggest that LTi are still present 300 days after clone 13 infection, lack of
LTi are unlikely to explain the failure of spleens to recover following LCMV clearance.
Moreover, despite their small size, the atrophied spleens have largely regained their normal
architecture. As chemokine gradients required for T cell/B cell segregation are dependent on
LTi, it appears unlikely that loss of these cells is the cause of splenic atrophy.
Lack of spleen recovery might also be due to defective chemokine/cytokine expression by
splenic stromal cells, aberrant generation of immune cells in the bone marrow/thymus, altered
homing of lymphocytes to non-lymphoid organs or possibly sustained autoreactive phenomena
induced by persistent viral infection. Indeed, splenic atrophy in humans is associated with celiac
disease and other autoimmune conditions such as SLE and Sjögren’s syndrome (226-231, 233).
It is not entirely clear whether splenic atrophy predisposes to autoimmunity, or vice versa, and
the mechanisms underlying splenic atrophy are not defined.
An association between splenic atrophy and viral infection has been shown for several viruses,
including SARS and H5N1 infection in humans (234, 235), as well as for coxsackievirus B3
(236), YN strain of parainfluenza virus (237), MHV68 (239), and TBEV (240) infections in
mice. In the mouse models, atrophic spleens were shown to be approximately 2-3 fold smaller
than uninfected controls in size (236, 237) and number of immune cells (236, 240) and the
kinetics of atrophy ranged from 5-14 days following infection (236, 237, 239, 240). In those
studies where the numbers of specific cell types in the spleen were assessed, it was shown that T
cell, B cell and non-B non-T cell subsets were affected (236, 239, 240). The exact mechanisms
underlying splenic atrophy or its causes were not clear in these studies, but were attributed to the
cytopathicity of the virus (234, 235), the effector functions of T cells (239) and cytokine
100
production (240). These studies did not investigate a potential role for NK cells in mediating
splenic atrophy, thus it is possible that similar to LCMV clone 13 infection, splenic atrophy
following these viral infections also depended in part on the presence of NK cells. In the
aforementioned studies, atrophy was irreversible as these viral infections ultimately killed their
hosts (236, 237, 240). MHV68 infection of IFNγR-/-
mice was an exception, in that similar to
LCMV clone 13 infection in our model, infection in these mice was not lethal, and mediated
irreversible splenic atrophy out to day 70 pi (239).
There is evidence that the spleen can regenerate following splenectomy and autotransplantantion
of spleen tissue in the peritoneum in mice and humans (251, 529, 530). The amount of spleen
tissue regenerated varies, but does not exceed that of the recipient’s own spleen (251, 531).
Regenerated splenic tissue appears histologically normal and can function in blood filtration,
however, the extent to which regenerated spleens protect from subsequent infection is not clear
(251, 532). The mechanisms regulating spleen tissue neogenesis have been investigated in
animal models, and although these are poorly defined, they are thought to involve the activity of
LT-educated endothelial organizer cells in the grafted spleen (533). Factors such as the age of
splenic tissue at the time of transplantation affects its ability to regenerate (533). Tan et al.
showed that grafting of whole-spleen capsules from D3 donors resulted in 83% (20/24) of the
grafts regenerating spleen tissue in recipient mice 4 weeks following transfer whereas only 38%
(6/16) of 8 week old adult spleen capsule grafts regenerated normal spleen tissue (533). It is
therefore possible that in our study, the atrophied spleens were too old to properly regenerate
following viral clearance. Other studies have shown that splenic tissue transplanted into
splenectomized mice regenerate to a larger size and contain more lymphoid tissue than spleen
grafts in sham-operated mice (534, 535). These studies suggest that during steady-state
conditions, there are factors secreted by the spleen or elsewhere that normally prevent
regeneration (251). This might also explain why the atrophied spleens induced by clone 13
infection had not returned to pre-infection weight by 300 dpi.
Taken together, these findings implicate NK cells in the mechanisms underlying pathological
splenic atrophy, and support a therapeutic role for NK cell depletion in the context of persistent
viral infection.
101
Chapter 4
Discussion and Future Directions
102
4.1 Overview
In this thesis, I have explored two aspects of innate immunity. In chapter 2, I demonstrated that
4-1BB and 4-1BBL interact consitutively on DCs and in chapter 3, I demonstrated a role for NK
cells in splenic atrophy in chronic LCMV infection. Here I discuss the implications of these
findings and possible future directions for the projects.
4.2 Role of 4-1BB/4-1BBL on DCs: What unique transcripts are induced by 4-1BB/4-1BBL
on DCs and does this impact secondary immune responses?
The finding that 4-1BB/4-1BBL interact on LPS-activated DCs (Fig. 4.1) raises the question of
the reason for such an interaction. What is the biological outcome of 4-1BB/4-1BBL interaction
for the DCs, and how does this interaction impact the interaction between DCs and other cell
types during an ongoing immune response in vivo? It is possible that 4-1BB/4-1BBL interaction
on DCs shuts down their ability to activate T cell responses by limiting the available 4-1BBL
signal for 4-1BB-expressing T cells, or by actively inhibiting pro-inflammatory signaling in the
DCs. Alternatively, this constitutive interaction could further enhance the stimulatory capabilities
of DCs before they encounter a T cell or during the DC-T cell interaction.
My studies in chapter 2 showed that 4-1BB/4-1BBL interaction on DCs was dispensable for the
primary response to influenza NP. As the conditions of immunization were chosen such that the
response was suboptimal, it seems unlikely that this negative result is due to the saturation of the
response. Although I tested the impact of 4-1BB/4-1BBL on DCs in the primary expansion of
influenza NP-specific CD8 T cells, it is conceivable that this interaction could be important in
programming a secondary response. The precedent for this type of experiment is the work of
Schoenberger et al. showing that T cells primed in the absence of CD4 T cell help, show
relatively normal primary expansion, but impaired secondary expansion (536), later attributed to
a role for CD4 T help during priming in inducing autocrine IL-2 production by the CD8 T cells
(537), which in turn prevented TRAIL upregulation during secondary antigen exposure, which
otherwise led to CD8 T cell death (538). To rule out this kind of scenario, one could check the
secondary response to influenza infection in mice immunized with DCs expressing influenza NP,
but lacking 4-1BB/4-1BBL. However, this scenario seems unlikely as two studies (436, 506)
showed that T cells primed in 4-1BBL-/-
mice by i.p. infection with influenza A/X31 and then
103
104
transferred to 4-1BBL-sufficient mice show normal secondary T cell expansion to a second
influenza infection with influenza A/PR8, suggesting that 4-1BBL is dispensable for
programming of CD8 T cell memory. Conflicting data on this point were obtained by Hendriks
et al. (448) using a different influenza strain, A/NT/60, which suggested that with a weak
primary response, 4-1BBL could be important in programming T cell memory. Thus an
outstanding question for the studies in chapter 2, is whether T cells primed with 4-1BB or 4-
1BBL-deficient DCs can mount an effective secondary T cell response. If not, this raises the
question of what 4-1BB/4-1BBL do on DCs during priming to allow full programming of CD8 T
cell memory.
Although we did not find a role for 4-1BB/4-1BBL interaction on DCs in priming influenza NP-
specific T cell responses, it is possible, indeed likely, that 4-1BB/4-1BBL interactions within the
DC compartment has a biological impact in some contexts. Thus, in order to take an unbiased
approach to decipher how 4-1BB and 4-1BBL regulate DC biology, future projects should
consider a high-throughput whole-genome screening approach, such as microarray technology
for gene expression studies in WT and deficient DCs. In this regard, it is worth considering
whether this should be done with LPS-activated BM-derived DCs ex vivo, or whether conditional
knockout mice lacking 4-1BB or 4-1BBL only on DCs, should be generated, so that the
consequences of this signal can be studied in vivo. Within the TNF family, conditional knockout
mice exist for TNF and CD70, and in the case of TNF, have been useful for determining which
TNF expressing cell types are mediators of inflammation in several models (539-541). However,
to date, 4-1BBL conditional knockout mice have not been generated.
4.3 4-1BB signaling in DCs
The 4-1BB signaling pathway in T cells has been well characterized and was briefly summarized
in the introduction section of this thesis (1.7.6). One of the outstanding questions is whether
TRAF recruitment and signaling have different consequences in different cell types. For
example, how do 4-1BB signals in a T cell compare to 4-1BB signals in a DC, and are these
important considerations for understanding the differential activity of 4-1BB on T cells versus
DCs. As 4-1BB regulates transcription factors such as NF-κB and MAPK-induced
transcritiptional regulators, it is likely that the genes induced by 4-1BB in different cell types
105
depend on the context. The molecular pathway of 4-1BB signaling in DCs has not been
investigated and is a topic for future studies.
4.4 Potential bidirectional signaling by 4-1BB and 4-1BBL on DCs
An interesting feature of the TNF superfamily is bidirectional signaling, that is, for some family
members, both receptor and ligand can signal (432, 542-546). When signals are transmitted
through the ligand, this is referred to as “reverse signaling”. Since DCs express both 4-1BB and
4-1BBL, it is not clear in our model whether regulation of DC biology will be mediated by 4-
1BB and/or 4-1BBL signaling.
4-1BBL reverse signaling has been described in T cells, monocytes, macrophages, B cells,
osteoclasts, and DCs (432, 544). For the most part, reverse signals have been shown to enhance
the activation and pro-inflammatory activity of these cell types, although in some cases, reverse
signaling mediates an inibitory signal. One caveat to these experiments is that it is often difficult
to interpret whether an antibody is mediating its effects by inducing reverse signals, blocking
receptor-ligand interactions, or both. Overall, studies evaluating the biological contribution of
reverse signaling during ongoing immune responses are lacking, as most studies have been
conducted in vitro using stimulating agents in cell lines and some primary cells. Another issue
here is the reagents, as well as their respective controls, used to stimulate 4-1BBL. A 4-1BB-Fc
protein that could also target Fc receptors is commonly used without the inclusion of 4-1BBL-
deficient control cells. This is an issue, because the oligomerization state of 4-1BB-Fc may be
different than that of the human IgG1 control that is often used (497, 547). Moving forward,
knockin-mutations in the cytoplasmic tail of 4-1BBL would aid in assessing the true ability of a
ligand to transmit signals.
Another area that requires further investigation is the precise mechanisms used by 4-1BBL to
transmit signals, and the molecules involved in the 4-1BBL signaling pathway. The cytoplasmic
tails of six TNF family members, 4-1BBL, CD70, TNF, FasL, CD40L and CD30L, contain one
or more consensus sequences for phosphorylation by the ubiquitously expressed kinase, casein
kinase I (CKI) (545). Recombinant CKI was shown to phosphorylate 4-1BBL (548), TNF (549)
and FasL (550), and mutagenesis experiments involving CKI inhibitors revealed that the CKI
motif is important for NFAT activation in FasL-mediated T cell costimulation (550).
106
Interestingly, 4-1BBL has been suggested to interact with molecules other than its traditional
receptor, 4-1BB, in mediating signals, including TLRs (440, 551), TNFR1 (552) and
TMEM126A (553). Taken together, evidence exists for bidirectional signaling during 4-1BB/4-
1BBL interactions, however, a demonstration that these signals have in vivo biological
significance is lacking.
4.5 Splenic atrophy following LCMV clone 13 infection
Chapter 3 identified NK cells as contributing to splenic atrophy following LCMV clone 13
infection, however, the mechanisms whereby NK cells mediate cell death were not delineated
(Fig. 4.2). My studies identified a potential role for the activating NK cell receptor, NKG2D, but
it is also possible that other activating NK cell receptors such as NCR1, are involved.
Alternatively, NK cell inhibitory ligands, such as MHC-I and Qa-1b which bind to the NK cell
inhibitory Ly49 and CD94/NKG2 receptors on NK cells, could be downregulated on these cells,
thereby making them susceptible to NK cell-mediated lysis. The upregulation of activating
ligands for NCR1 and downregulation of inhibitory ligands MHC-I and Qa-1b were recently
identified as mechanisms that render anti-viral T cells susceptible to NK cell killing in the
absence of early type I IFN signaling during LCMV WE infection (326, 327). The inhibitory
receptor 2B4 on NK cells has also been shown to be involved in NK cell killing of activated T
cells during LCMV clone 13 infection (325). Future studies should investigate further the NK
cell receptor-ligand interactions mediating splenic atrophy during LCMV clone 13 infection.
To look at a potential involvement of the activating receptor NCR1, future studies can utilize an
NCR1-IgG fusion protein, which detects NCR1 ligands (139), to assess the expression of NCR1
ligands at later time points following Cl13 infection. An NCR1 blocking antibody and NCR1-
deficient mice (554) are also available to test the impact of NCR1/NCR1 ligand interactions in
vivo. The expression of inhibitory ligands such as MHC-I, Qa-1b, and CD48 following Cl13
infection can be detected by using specifc antibodies for flow cytometry. β-2 microglobulin-
deficient (β-2m-/-
) mice (555), which lack surface MHC class I expression, and CD48-/-
mice
(556) are also available. Splenocytes from these mice could be used in ex vivo or in vivo NK cell
cytotoxicity assays to assess whether NK cells require detection of these ligands for killing. Ex
vivo, one can measure the apoptosis of cells cocultured with NK cells isolated from LCMV-
infected mice (19-21+ dpi) by flow cytometry using reagents specific for apoptotic cells
107
108
such as an antibody to Annexin-V and 7-AAD viability staining solution (326). In vivo, CFSE-
labelled target cells can be transferred into LCMV-infected mice (19-21+ dpi) and tracked for
short periods following transfer by CFSE expression. To further look at the expression of
NKG2D ligands, an NKG2D-human IgG fusion protein (557), which detects MULT1, the
isoforms of Rae-1, and H60, can be used. The involvement of NKG2D can also be verified by
using an NKG2D blocking antibody (558, 559) in vivo or Klrk1-/-
mice, which are deficient in
NKG2D.
The caveat to using mice that have a deficit in NK cell numbers, e.g. Nfil3-/-
(E4bp4-/-
) mice
(560), in the Cl13 splenic atrophy model is that Cl13 infection of these mice is predicted to not
cause a persistent infection due to an increased number of functional CD8 T cells mediating viral
clearance. Indeed, infection of Nfil3-/-
mice with LCMV WE resulted in double the frequency of
IFNγ-producing CD8 T cells 6 dpi when compared to WT mice (322). Thus, Cl13 infection of
NK cell-deficient mice could resemble an acute LCMV infection, which I showed in this model
does not result in splenic atrophy.
The findings in chapter 3 of this thesis do not definitively identify NK cells as the only
mechanism causing splenic atrophy following LCMV clone 13 infection as NK cell depletion
did not fully abrogate splenic contraction below pre-infection spleen weight. It is not clear,
however, whether the lack of complete reversal of splenic atrophy by NK cell depletion is
because NK cells are not the only mechanism involved or whether the depletion is incomplete. It
is possible that other cell types such as cytotoxic CD8 T cells or macrophages contribute to the
dramatic loss of cells. Indeed, in a model of splenic atrophy following MHV68 infection of
IFNγR-/-
mice, CD8 T cell depletion abrogated splenic atrophy (239). CD8 T cells also express
NKG2D and are involved in the killing of NKG2D ligand-expressing cells. Depleting CD8 T
cells or possibly macrophages at the onset of splenic atrophy would allow us to evaluate the
potential contribution of these cell types.
It should be noted that NKT cells also express NK1.1 and could be depleted by the high dose of
anti-NK1.1 (clone PK136) antibody used in this study. NKT cells are non-conventional αβ T
cells that express molecules associated with the NK cell lineage and are restricted by the
monomorphic MHC class-I-like molecule CD1d that presents self and exogenous glycolipids
(561). Type I NKT cells or invariant NKT cells (iNKT) cells express an invariant TCRα chain
109
encoded by Vα14-Jα18 genes in mice and Vα24-Jα18 genes in humans and a restricted set of
TCRβ chains (Vβ2, Vβ7 and Vβ8 in mice and Vβ11 in humans), whereas type II NKT cells
express more diverse TCR Vα chains (561). Following activation, NKT cells produce large
amounts of IFNγ and IL-4, which influence a wide array of immune responses, including tumor
surveillance, maintenance of self-tolerance and anti-microbial defenses (561). LCMV Armstrong
infection of Vα14 transgenic mice, which contain an elevated frequency of iNKT cells, and
treatment of C57BL/6 mice with the iNKT cell agonist α-galactoside ceramide (α-GalCer) during
Armstrong infection revealed a role for NKT cells in controlling viral load in the liver and
pancreas, but not at early time points (up to 4 dpi) in the spleen (562). Compared to control mice,
viral load was about 1 log lower in Armstrong-infected Vα14 transgenic mice 6-8 dpi, suggesting
a possible delayed role for NKT cells in controlling virus in the spleen (562). NKT cells were
shown to be dispensable for NK cell immunoregulatory function during LCMV Cl13 infection as
NK cell depletion of Cl13-infected CD1d-/-
mice, which lack NKT cells, enhanced LCMV-
specific T cell responses and reduced viral load, similar to that in WT mice (323).
To evaluate a potential role of NKT cells in the LCMV clone 13 splenic atrophy model, the
frequency of NKT cells before and after anti-NK1.1 treatment should first be determined using
NKT tetramers. The frequency of other NK1.1-expressing cell types such as activated CD8 T
cells and γδ T cells following anti-NK1.1 depletion should also be assessed. A role for NKT cells
and γδ T cells can be further evaluated using CD1d-/-
and TCRδ-/-
mice.
4.6 Can the spleen regenerate following splenic atrophy?
One question that this study raises is whether during steady state conditions, the spleen fully
regenerates following splenic atrophy, and if so, how. If the spleen does not regenerate, then why
not?
Splenic regeneration or splenosis (autotransplantation of splenic tissue) is a normal process that
can occur following splenectomy due to trauma or pathology of the spleen. This occurs when
cells from a damaged spleen seed elsewhere on the peritoneal surface and grow into nodules of
differentiated splenic tissue with varying size. They are supplied by newly formed arteries that
penetrate the capsule (563). Patients that undergo splenectomy due to trauma rather than
hematological disorders are more likely to experience splenic regeneration due to the increased
110
likelihood of dissipation of splenic tissue, which favours autotransplantation, and therefore these
patients have an overall lower risk of subsequent infection and death. The rate of splenic
regeneration following traumatic splenectomy is as high as 66% (564). Another factor reducing
the risk of infection following splenectomy is the hypertrophy of functional splenuncli, which are
accessory spleens resulting from the incomplete fusion of separated fetal spleen tissue
originating from the dorsal mesogastrium (565). Accessory spleens are reported to have an
incidence of over 10% (566).
Splenic regeneration also occurs following manual autotransplantation of splenic tissue.
Following splenectomy in humans, the removed spleen is cut into thin slices and re-implanted
into omental pouches, in order for it to regenerate (530). The specific cells and molecules
regulating spleen tissue neogenesis have been investigated in animal models, although these are
poorly defined. It is known that following spleen transplantation, the process of tissue
regeneration involves tissue necrosis within 4 days of autotransplantation, regeneration of outer
tissue layer and differentiation of connective tissue into splenic reticular cells, vascular re-growth
within 8 days with lymphocytes around primitive vessels, and white pulp differentiation within 5
weeks. Remarkably, the newly generated spleen appears histologically normal, and red and white
pulp areas are clearly distinct (251).
A recent study showed that mouse splenic tissue regeneration following transplantation of
neonatal spleen capsule tissue into the renal subcapsular space depended only on the presence of
stromal cells in the transplant and on the LT pathway. RAG-/-
spleen capsules were capable of
regenerating following transplantation, whereas LTα-/-
spleen capsules failed to induce tissue
regeneration. These investigators point to the activity of LT-educated endothelial cell organizers
in orchestrating splenic regeneration (533).
A major question in this field is whether the function of these newly generated spleens is
comparable to a normal spleen. Reported post-operative findings include a reduction in
thrombocytosis, increased levels of IgM, and loss of Howell-Jolly bodies and red-pitted cells
(532). Whether these regenerated spleens are able to protect from subsequent bacterial infection
is less clear-cut, but the data does suggest that the volume of regenerated splenic tissue seems to
correlate with protection against post-splenectomy infection (567, 568). It is noteworthy that
factors including the age of the transplanted splenic tissue, the location to which it is
111
transplanted, and the time between removal of tissue and transplantation are all factors
determining the ability of the splenic tissue to regenerate, whereas age of the donor seems not to
be an important factor.
Although it is clear that the spleen can regenerate following disorder/trauma and splenectomy
and autotransplantation, it is not clear whether it recovers from splenic atrophy. Splenic atrophy
was shown to be irreversible in a study of celiac disease patients. Necropsy studies in adult celiac
disease show the spleen to become thickly encapsulated with fibrous tissue, and the red pulp to
be replaced with fibrous tissue, processes which are not easily reversible (231). Of interest,
transplantation studies showed that splenic fragments do not grow as well if the spleen is also
present, and that there is a limit to the size to which splenic fragments will grow, a size not
exceeding that of the original spleen. This suggests that the spleen or an intermediary system
produces negative feedback inhibitors of growth that normally prevent regeneration (251, 531,
534, 569). This might explain why the spleen may not normally regenerate following atrophy.
In the LCMV clone 13-induced splenic atrophy model discussed in chapter 3 of this thesis,
spleens did not recover to pre-infection weight out to day 300 pi, which was the last time point
assessed. The reason for this is not defined, but might involve the age of the mice when atrophy
occurs and when virus is cleared. Tan et al. showed that grafting of whole-spleen capsules from
D3 donors resulted in 83% (20/24) of the grafts regenerating spleen tissue in recipient mice 4
weeks following transfer whereas only 38% (6/16) of adult spleen capsule grafts regenerated
normal spleen tissue (533). Therefore, as briefly mentioned above, the age of splenic tissue does
factor into its ability to regenerate. It is possible that the spleens from Cl13-infected mice are too
old to properly regenerate following viral clearance. To determine whether the spleen ever fully
regenerates to pre-infection weight following atrophy in this model, LCMV clone 13-infected
mice should be housed in specific pathogen-free conditions for at least 2 years.
4.7 Conclusion
My studies in chapter 2 have shown that 4-1BB and 4-1BBL constitutively interact on LPS-
activated DCs, adding to the growing body of evidence that receptors and ligands of the TNF
family have the ability to interact on multiple cell types, including interacting within a single cell
type. More work is required to understand the functional role of this interaction. In chapter 3, I
112
revealed remarkably long-term splenic atrophy following LCMV clone 13 infection, months
after virus can no longer be detected. I also showed a role for NK cells in mediating splenic
atrophy at late stages following clone 13 infection. This raises the issue of whether NK cells are
prominent in human conditions with splenic atrophy, including chronic infections such as HIV.
113
Chapter 5
References
114
References
1. Oldstone MB. 2013. Lessons learned and concepts formed from study of the pathogenesis
of the two negative-strand viruses lymphocytic choriomeningitis and influenza. Proc Natl
Acad Sci U S A 110: 4180-3
2. Virgin HW, Wherry EJ, Ahmed R. 2009. Redefining chronic viral infection. Cell 138:
30-50
3. Akira S, Uematsu S, Takeuchi O. 2006. Pathogen recognition and innate immunity. Cell
124: 783-801
4. Takeuchi O, Akira S. 2009. Innate immunity to virus infection. Immunol Rev 227: 75-86
5. Barton GM, Kagan JC. 2009. A cell biological view of Toll-like receptor function:
regulation through compartmentalization. Nat Rev Immunol 9: 535-42
6. Kawai T, Akira S. 2010. The role of pattern-recognition receptors in innate immunity:
update on Toll-like receptors. Nat Immunol 11: 373-84
7. Kawai T, Akira S. 2009. The roles of TLRs, RLRs and NLRs in pathogen recognition. Int
Immunol 21: 317-37
8. Loo YM, Gale M, Jr. 2011. Immune signaling by RIG-I-like receptors. Immunity 34: 680-
92
9. Paz S, Sun Q, Nakhaei P, Romieu-Mourez R, Goubau D, Julkunen I, Lin R, Hiscott J.
2006. Induction of IRF-3 and IRF-7 phosphorylation following activation of the RIG-I
pathway. Cell Mol Biol (Noisy-le-grand) 52: 17-28
10. Dinarello CA. 2009. Immunological and inflammatory functions of the interleukin-1
family. Annu Rev Immunol 27: 519-50
11. Schroder K, Tschopp J. 2010. The inflammasomes. Cell 140: 821-32
12. Fogg DK, Sibon C, Miled C, Jung S, Aucouturier P, Littman DR, Cumano A, Geissmann
F. 2006. A clonogenic bone marrow progenitor specific for macrophages and dendritic
cells. Science 311: 83-7
13. Jakubzick C, Bogunovic M, Bonito AJ, Kuan EL, Merad M, Randolph GJ. 2008. Lymph-
migrating, tissue-derived dendritic cells are minor constituents within steady-state lymph
nodes. J Exp Med 205: 2839-50
14. Naik SH, Metcalf D, van Nieuwenhuijze A, Wicks I, Wu L, O'Keeffe M, Shortman K.
2006. Intrasplenic steady-state dendritic cell precursors that are distinct from monocytes.
Nat Immunol 7: 663-71
15. Varol C, Landsman L, Fogg DK, Greenshtein L, Gildor B, Margalit R, Kalchenko V,
Geissmann F, Jung S. 2007. Monocytes give rise to mucosal, but not splenic,
conventional dendritic cells. J Exp Med 204: 171-80
16. Liu K, Nussenzweig MC. 2010. Origin and development of dendritic cells. Immunol Rev
234: 45-54
17. Ginhoux F, Liu K, Helft J, Bogunovic M, Greter M, Hashimoto D, Price J, Yin N,
Bromberg J, Lira SA, Stanley ER, Nussenzweig M, Merad M. 2009. The origin and
development of nonlymphoid tissue CD103+ DCs. J Exp Med 206: 3115-30
18. Kabashima K, Banks TA, Ansel KM, Lu TT, Ware CF, Cyster JG. 2005. Intrinsic
lymphotoxin-beta receptor requirement for homeostasis of lymphoid tissue dendritic
cells. Immunity 22: 439-50
19. Liu K, Waskow C, Liu X, Yao K, Hoh J, Nussenzweig M. 2007. Origin of dendritic cells
in peripheral lymphoid organs of mice. Nat Immunol 8: 578-83
115
20. Maraskovsky E, Brasel K, Teepe M, Roux ER, Lyman SD, Shortman K, McKenna HJ.
1996. Dramatic increase in the numbers of functionally mature dendritic cells in Flt3
ligand-treated mice: multiple dendritic cell subpopulations identified. J Exp Med 184:
1953-62
21. Waskow C, Liu K, Darrasse-Jeze G, Guermonprez P, Ginhoux F, Merad M, Shengelia T,
Yao K, Nussenzweig M. 2008. The receptor tyrosine kinase Flt3 is required for dendritic
cell development in peripheral lymphoid tissues. Nat Immunol 9: 676-83
22. Kamath AT, Henri S, Battye F, Tough DF, Shortman K. 2002. Developmental kinetics
and lifespan of dendritic cells in mouse lymphoid organs. Blood 100: 1734-41
23. Idoyaga J, Suda N, Suda K, Park CG, Steinman RM. 2009. Antibody to Langerin/CD207
localizes large numbers of CD8alpha+ dendritic cells to the marginal zone of mouse
spleen. Proc Natl Acad Sci U S A 106: 1524-9
24. Belz GT, Behrens GM, Smith CM, Miller JF, Jones C, Lejon K, Fathman CG, Mueller
SN, Shortman K, Carbone FR, Heath WR. 2002. The CD8alpha(+) dendritic cell is
responsible for inducing peripheral self-tolerance to tissue-associated antigens. J Exp
Med 196: 1099-104
25. den Haan JM, Lehar SM, Bevan MJ. 2000. CD8(+) but not CD8(-) dendritic cells cross-
prime cytotoxic T cells in vivo. J Exp Med 192: 1685-96
26. Iyoda T, Shimoyama S, Liu K, Omatsu Y, Akiyama Y, Maeda Y, Takahara K, Steinman
RM, Inaba K. 2002. The CD8+ dendritic cell subset selectively endocytoses dying cells
in culture and in vivo. J Exp Med 195: 1289-302
27. Shortman K, Heath WR. 2010. The CD8+ dendritic cell subset. Immunol Rev 234: 18-31
28. Dudziak D, Kamphorst AO, Heidkamp GF, Buchholz VR, Trumpfheller C, Yamazaki S,
Cheong C, Liu K, Lee HW, Park CG, Steinman RM, Nussenzweig MC. 2007.
Differential antigen processing by dendritic cell subsets in vivo. Science 315: 107-11
29. Kamphorst AO, Guermonprez P, Dudziak D, Nussenzweig MC. 2010. Route of antigen
uptake differentially impacts presentation by dendritic cells and activated monocytes. J
Immunol 185: 3426-35
30. Shortman K, Liu YJ. 2002. Mouse and human dendritic cell subtypes. Nat Rev Immunol
2: 151-61
31. Allan RS, Waithman J, Bedoui S, Jones CM, Villadangos JA, Zhan Y, Lew AM,
Shortman K, Heath WR, Carbone FR. 2006. Migratory dendritic cells transfer antigen to
a lymph node-resident dendritic cell population for efficient CTL priming. Immunity 25:
153-62
32. Ohl L, Mohaupt M, Czeloth N, Hintzen G, Kiafard Z, Zwirner J, Blankenstein T,
Henning G, Forster R. 2004. CCR7 governs skin dendritic cell migration under
inflammatory and steady-state conditions. Immunity 21: 279-88
33. Itano AA, Jenkins MK. 2003. Antigen presentation to naive CD4 T cells in the lymph
node. Nat Immunol 4: 733-9
34. Dzionek A, Fuchs A, Schmidt P, Cremer S, Zysk M, Miltenyi S, Buck DW, Schmitz J.
2000. BDCA-2, BDCA-3, and BDCA-4: three markers for distinct subsets of dendritic
cells in human peripheral blood. J Immunol 165: 6037-46
35. Bachem A, Guttler S, Hartung E, Ebstein F, Schaefer M, Tannert A, Salama A,
Movassaghi K, Opitz C, Mages HW, Henn V, Kloetzel PM, Gurka S, Kroczek RA. 2010.
Superior antigen cross-presentation and XCR1 expression define human
116
CD11c+CD141+ cells as homologues of mouse CD8+ dendritic cells. J Exp Med 207:
1273-81
36. Crozat K, Guiton R, Contreras V, Feuillet V, Dutertre CA, Ventre E, Vu Manh TP,
Baranek T, Storset AK, Marvel J, Boudinot P, Hosmalin A, Schwartz-Cornil I, Dalod M.
2010. The XC chemokine receptor 1 is a conserved selective marker of mammalian cells
homologous to mouse CD8alpha+ dendritic cells. J Exp Med 207: 1283-92
37. Robbins SH, Walzer T, Dembele D, Thibault C, Defays A, Bessou G, Xu H, Vivier E,
Sellars M, Pierre P, Sharp FR, Chan S, Kastner P, Dalod M. 2008. Novel insights into the
relationships between dendritic cell subsets in human and mouse revealed by genome-
wide expression profiling. Genome Biol 9: R17
38. Crozat K, Guiton R, Guilliams M, Henri S, Baranek T, Schwartz-Cornil I, Malissen B,
Dalod M. 2010. Comparative genomics as a tool to reveal functional equivalences
between human and mouse dendritic cell subsets. Immunol Rev 234: 177-98
39. Perussia B, Fanning V, Trinchieri G. 1985. A leukocyte subset bearing HLA-DR antigens
is responsible for in vitro alpha interferon production in response to viruses. Nat Immun
Cell Growth Regul 4: 120-37
40. Cella M, Jarrossay D, Facchetti F, Alebardi O, Nakajima H, Lanzavecchia A, Colonna M.
1999. Plasmacytoid monocytes migrate to inflamed lymph nodes and produce large
amounts of type I interferon. Nat Med 5: 919-23
41. Siegal FP, Kadowaki N, Shodell M, Fitzgerald-Bocarsly PA, Shah K, Ho S, Antonenko
S, Liu YJ. 1999. The nature of the principal type 1 interferon-producing cells in human
blood. Science 284: 1835-7
42. Colonna M, Trinchieri G, Liu YJ. 2004. Plasmacytoid dendritic cells in immunity. Nat
Immunol 5: 1219-26
43. O'Keeffe M, Hochrein H, Vremec D, Scott B, Hertzog P, Tatarczuch L, Shortman K.
2003. Dendritic cell precursor populations of mouse blood: identification of the murine
homologues of human blood plasmacytoid pre-DC2 and CD11c+ DC1 precursors. Blood
101: 1453-9
44. Crotty S. 2011. Follicular helper CD4 T cells (TFH). Annu Rev Immunol 29: 621-63
45. Granucci F, Foti M, Ricciardi-Castagnoli P. 2005. Dendritic cell biology. Adv Immunol
88: 193-233
46. Zygmunt B, Veldhoen M. 2011. T helper cell differentiation more than just cytokines.
Adv Immunol 109: 159-96
47. Maldonado-Lopez R, De Smedt T, Michel P, Godfroid J, Pajak B, Heirman C,
Thielemans K, Leo O, Urbain J, Moser M. 1999. CD8alpha+ and CD8alpha- subclasses
of dendritic cells direct the development of distinct T helper cells in vivo. J Exp Med 189:
587-92
48. Pulendran B, Smith JL, Caspary G, Brasel K, Pettit D, Maraskovsky E, Maliszewski CR.
1999. Distinct dendritic cell subsets differentially regulate the class of immune response
in vivo. Proc Natl Acad Sci U S A 96: 1036-41
49. Shen L, Rock KL. 2006. Priming of T cells by exogenous antigen cross-presented on
MHC class I molecules. Curr Opin Immunol 18: 85-91
50. Banchereau J, Briere F, Caux C, Davoust J, Lebecque S, Liu YJ, Pulendran B, Palucka K.
2000. Immunobiology of dendritic cells. Annu Rev Immunol 18: 767-811
51. Manicassamy S, Pulendran B. 2011. Dendritic cell control of tolerogenic responses.
Immunol Rev 241: 206-27
117
52. Bevan MJ. 2004. Helping the CD8(+) T-cell response. Nat Rev Immunol 4: 595-602
53. Bennett SR, Carbone FR, Karamalis F, Flavell RA, Miller JF, Heath WR. 1998. Help for
cytotoxic-T-cell responses is mediated by CD40 signalling. Nature 393: 478-80
54. Ridge JP, Di Rosa F, Matzinger P. 1998. A conditioned dendritic cell can be a temporal
bridge between a CD4+ T-helper and a T-killer cell. Nature 393: 474-8
55. Schoenberger SP, Toes RE, van der Voort EI, Offringa R, Melief CJ. 1998. T-cell help
for cytotoxic T lymphocytes is mediated by CD40-CD40L interactions. Nature 393: 480-
3
56. Summers deLuca L, Ng D, Gao Y, Wortzman ME, Watts TH, Gommerman JL. 2011.
LTbetaR signaling in dendritic cells induces a type I IFN response that is required for
optimal clonal expansion of CD8+ T cells. Proc Natl Acad Sci U S A 108: 2046-51
57. Ng D, Gommerman JL. 2013. The Regulation of Immune Responses by DC Derived
Type I IFN. Front Immunol 4: 94
58. Bergtold A, Desai DD, Gavhane A, Clynes R. 2005. Cell surface recycling of internalized
antigen permits dendritic cell priming of B cells. Immunity 23: 503-14
59. Jego G, Pascual V, Palucka AK, Banchereau J. 2005. Dendritic cells control B cell
growth and differentiation. Curr Dir Autoimmun 8: 124-39
60. Qi H, Egen JG, Huang AY, Germain RN. 2006. Extrafollicular activation of lymph node
B cells by antigen-bearing dendritic cells. Science 312: 1672-6
61. Batista FD, Harwood NE. 2009. The who, how and where of antigen presentation to B
cells. Nat Rev Immunol 9: 15-27
62. Freer G, Matteucci D. 2009. Influence of dendritic cells on viral pathogenicity. PLoS
Pathog 5: e1000384
63. Geijtenbeek TB, van Kooyk Y. 2003. Pathogens target DC-SIGN to influence their fate
DC-SIGN functions as a pathogen receptor with broad specificity. APMIS 111: 698-714
64. de Witte L, Nabatov A, Geijtenbeek TB. 2008. Distinct roles for DC-SIGN+-dendritic
cells and Langerhans cells in HIV-1 transmission. Trends Mol Med 14: 12-9
65. Marzi A, Moller P, Hanna SL, Harrer T, Eisemann J, Steinkasserer A, Becker S,
Baribaud F, Pohlmann S. 2007. Analysis of the interaction of Ebola virus glycoprotein
with DC-SIGN (dendritic cell-specific intercellular adhesion molecule 3-grabbing
nonintegrin) and its homologue DC-SIGNR. J Infect Dis 196 Suppl 2: S237-46
66. Mahnke K, Guo M, Lee S, Sepulveda H, Swain SL, Nussenzweig M, Steinman RM.
2000. The dendritic cell receptor for endocytosis, DEC-205, can recycle and enhance
antigen presentation via major histocompatibility complex class II-positive lysosomal
compartments. J Cell Biol 151: 673-84
67. Bozzacco L, Trumpfheller C, Siegal FP, Mehandru S, Markowitz M, Carrington M,
Nussenzweig MC, Piperno AG, Steinman RM. 2007. DEC-205 receptor on dendritic
cells mediates presentation of HIV gag protein to CD8+ T cells in a spectrum of human
MHC I haplotypes. Proc Natl Acad Sci U S A 104: 1289-94
68. Lahoud MH, Ahmet F, Zhang JG, Meuter S, Policheni AN, Kitsoulis S, Lee CN,
O'Keeffe M, Sullivan LC, Brooks AG, Berry R, Rossjohn J, Mintern JD, Vega-Ramos J,
Villadangos JA, Nicola NA, Nussenzweig MC, Stacey KJ, Shortman K, Heath WR,
Caminschi I. 2012. DEC-205 is a cell surface receptor for CpG oligonucleotides. Proc
Natl Acad Sci U S A 109: 16270-5
69. Shrimpton RE, Butler M, Morel AS, Eren E, Hue SS, Ritter MA. 2009. CD205 (DEC-
205): a recognition receptor for apoptotic and necrotic self. Mol Immunol 46: 1229-39
118
70. Zhang SS, Park CG, Zhang P, Bartra SS, Plano GV, Klena JD, Skurnik M, Hinnebusch
BJ, Chen T. 2008. Plasminogen activator Pla of Yersinia pestis utilizes murine DEC-205
(CD205) as a receptor to promote dissemination. J Biol Chem 283: 31511-21
71. Bousarghin L, Hubert P, Franzen E, Jacobs N, Boniver J, Delvenne P. 2005. Human
papillomavirus 16 virus-like particles use heparan sulfates to bind dendritic cells and
colocalize with langerin in Langerhans cells. J Gen Virol 86: 1297-305
72. Da Silva DM, Fausch SC, Verbeek JS, Kast WM. 2007. Uptake of human papillomavirus
virus-like particles by dendritic cells is mediated by Fcgamma receptors and contributes
to acquisition of T cell immunity. J Immunol 178: 7587-97
73. de Witte L, Nabatov A, Pion M, Fluitsma D, de Jong MA, de Gruijl T, Piguet V, van
Kooyk Y, Geijtenbeek TB. 2007. Langerin is a natural barrier to HIV-1 transmission by
Langerhans cells. Nat Med 13: 367-71
74. Di Pucchio T, Chatterjee B, Smed-Sorensen A, Clayton S, Palazzo A, Montes M, Xue Y,
Mellman I, Banchereau J, Connolly JE. 2008. Direct proteasome-independent cross-
presentation of viral antigen by plasmacytoid dendritic cells on major histocompatibility
complex class I. Nat Immunol 9: 551-7
75. Smit JJ, Lindell DM, Boon L, Kool M, Lambrecht BN, Lukacs NW. 2008. The balance
between plasmacytoid DC versus conventional DC determines pulmonary immunity to
virus infections. PLoS One 3: e1720
76. Sevilla N, McGavern DB, Teng C, Kunz S, Oldstone MB. 2004. Viral targeting of
hematopoietic progenitors and inhibition of DC maturation as a dual strategy for immune
subversion. J Clin Invest 113: 737-45
77. Lockridge KM, Zhou SS, Kravitz RH, Johnson JL, Sawai ET, Blewett EL, Barry PA.
2000. Primate cytomegaloviruses encode and express an IL-10-like protein. Virology
268: 272-80
78. Barends M, de Rond LG, Dormans J, van Oosten M, Boelen A, Neijens HJ, Osterhaus
AD, Kimman TG. 2004. Respiratory syncytial virus, pneumonia virus of mice, and
influenza A virus differently affect respiratory allergy in mice. Clin Exp Allergy 34: 488-
96
79. Bueno SM, Gonzalez PA, Pacheco R, Leiva ED, Cautivo KM, Tobar HE, Mora JE, Prado
CE, Zuniga JP, Jimenez J, Riedel CA, Kalergis AM. 2008. Host immunity during RSV
pathogenesis. Int Immunopharmacol 8: 1320-9
80. Frank I, Piatak M, Jr., Stoessel H, Romani N, Bonnyay D, Lifson JD, Pope M. 2002.
Infectious and whole inactivated simian immunodeficiency viruses interact similarly with
primate dendritic cells (DCs): differential intracellular fate of virions in mature and
immature DCs. J Virol 76: 2936-51
81. Freer G, Matteucci D, Mazzetti P, Tarabella F, Catalucci V, Bendinelli M. 2007. Effects
of feline immunodeficiency virus on feline monocyte-derived dendritic cells infected by
spinoculation. J Gen Virol 88: 2574-82
82. Izquierdo-Useros N, Blanco J, Erkizia I, Fernandez-Figueras MT, Borras FE, Naranjo-
Gomez M, Bofill M, Ruiz L, Clotet B, Martinez-Picado J. 2007. Maturation of blood-
derived dendritic cells enhances human immunodeficiency virus type 1 capture and
transmission. J Virol 81: 7559-70
83. Palucka K, Banchereau J. 2012. Cancer immunotherapy via dendritic cells. Nat Rev
Cancer 12: 265-77
119
84. Palucka K, Banchereau J. 2013. Dendritic-cell-based therapeutic cancer vaccines.
Immunity 39: 38-48
85. Lapenta C, Santini SM, Logozzi M, Spada M, Andreotti M, Di Pucchio T, Parlato S,
Belardelli F. 2003. Potent immune response against HIV-1 and protection from virus
challenge in hu-PBL-SCID mice immunized with inactivated virus-pulsed dendritic cells
generated in the presence of IFN-alpha. J Exp Med 198: 361-7
86. Lu W, Wu X, Lu Y, Guo W, Andrieu JM. 2003. Therapeutic dendritic-cell vaccine for
simian AIDS. Nat Med 9: 27-32
87. Yoshida A, Tanaka R, Murakami T, Takahashi Y, Koyanagi Y, Nakamura M, Ito M,
Yamamoto N, Tanaka Y. 2003. Induction of protective immune responses against R5
human immunodeficiency virus type 1 (HIV-1) infection in hu-PBL-SCID mice by
intrasplenic immunization with HIV-1-pulsed dendritic cells: possible involvement of a
novel factor of human CD4(+) T-cell origin. J Virol 77: 8719-28
88. Aline F, Brand D, Bout D, Pierre J, Fouquenet D, Verrier B, Dimier-Poisson I. 2007.
Generation of specific Th1 and CD8+ T-cell responses by immunization with mouse
CD8+ dendritic cells loaded with HIV-1 viral lysate or envelope glycoproteins. Microbes
Infect 9: 536-43
89. Garcia F, Climent N, Guardo AC, Gil C, Leon A, Autran B, Lifson JD, Martinez-Picado
J, Dalmau J, Clotet B, Gatell JM, Plana M, Gallart T. 2013. A dendritic cell-based
vaccine elicits T cell responses associated with control of HIV-1 replication. Sci Transl
Med 5: 166ra2
90. Spits H, Artis D, Colonna M, Diefenbach A, Di Santo JP, Eberl G, Koyasu S, Locksley
RM, McKenzie AN, Mebius RE, Powrie F, Vivier E. 2013. Innate lymphoid cells--a
proposal for uniform nomenclature. Nat Rev Immunol 13: 145-9
91. Vivier E, Tomasello E, Baratin M, Walzer T, Ugolini S. 2008. Functions of natural killer
cells. Nat Immunol 9: 503-10
92. Yokoyama WM, Kim S, French AR. 2004. The dynamic life of natural killer cells. Annu
Rev Immunol 22: 405-29
93. Di Santo JP. 2006. Natural killer cell developmental pathways: a question of balance.
Annu Rev Immunol 24: 257-86
94. Williams NS, Klem J, Puzanov IJ, Sivakumar PV, Bennett M, Kumar V. 1999.
Differentiation of NK1.1+, Ly49+ NK cells from flt3+ multipotent marrow progenitor
cells. J Immunol 163: 2648-56
95. Schmitt TM, Zuniga-Pflucker JC. 2002. Induction of T cell development from
hematopoietic progenitor cells by delta-like-1 in vitro. Immunity 17: 749-56
96. Lehar SM, Dooley J, Farr AG, Bevan MJ. 2005. Notch ligands Delta 1 and Jagged1
transmit distinct signals to T-cell precursors. Blood 105: 1440-7
97. Kim S, Iizuka K, Kang HS, Dokun A, French AR, Greco S, Yokoyama WM. 2002. In
vivo developmental stages in murine natural killer cell maturation. Nat Immunol 3: 523-8
98. Anfossi N, Andre P, Guia S, Falk CS, Roetynck S, Stewart CA, Breso V, Frassati C,
Reviron D, Middleton D, Romagne F, Ugolini S, Vivier E. 2006. Human NK cell
education by inhibitory receptors for MHC class I. Immunity 25: 331-42
99. Chalifour A, Scarpellino L, Back J, Brodin P, Devevre E, Gros F, Levy F, Leclercq G,
Hoglund P, Beermann F, Held W. 2009. A Role for cis Interaction between the Inhibitory
Ly49A receptor and MHC class I for natural killer cell education. Immunity 30: 337-47
120
100. Fernandez NC, Treiner E, Vance RE, Jamieson AM, Lemieux S, Raulet DH. 2005. A
subset of natural killer cells achieves self-tolerance without expressing inhibitory
receptors specific for self-MHC molecules. Blood 105: 4416-23
101. Johansson S, Johansson M, Rosmaraki E, Vahlne G, Mehr R, Salmon-Divon M,
Lemonnier F, Karre K, Hoglund P. 2005. Natural killer cell education in mice with single
or multiple major histocompatibility complex class I molecules. J Exp Med 201: 1145-55
102. Kim S, Poursine-Laurent J, Truscott SM, Lybarger L, Song YJ, Yang L, French AR,
Sunwoo JB, Lemieux S, Hansen TH, Yokoyama WM. 2005. Licensing of natural killer
cells by host major histocompatibility complex class I molecules. Nature 436: 709-13
103. Sun JC, Lanier LL. 2011. NK cell development, homeostasis and function: parallels with
CD8(+) T cells. Nat Rev Immunol 11: 645-57
104. Brady J, Carotta S, Thong RP, Chan CJ, Hayakawa Y, Smyth MJ, Nutt SL. 2010. The
interactions of multiple cytokines control NK cell maturation. J Immunol 185: 6679-88
105. Loza MJ, Peters SP, Zangrilli JG, Perussia B. 2002. Distinction between IL-13+ and IFN-
gamma+ natural killer cells and regulation of their pool size by IL-4. Eur J Immunol 32:
413-23
106. Loza MJ, Zamai L, Azzoni L, Rosati E, Perussia B. 2002. Expression of type 1
(interferon gamma) and type 2 (interleukin-13, interleukin-5) cytokines at distinct stages
of natural killer cell differentiation from progenitor cells. Blood 99: 1273-81
107. Perussia B, Chen Y, Loza MJ. 2005. Peripheral NK cell phenotypes: multiple changing
of faces of an adapting, developing cell. Mol Immunol 42: 385-95
108. Di Santo JP, Vosshenrich CA. 2006. Bone marrow versus thymic pathways of natural
killer cell development. Immunol Rev 214: 35-46
109. Ma A, Koka R, Burkett P. 2006. Diverse functions of IL-2, IL-15, and IL-7 in lymphoid
homeostasis. Annu Rev Immunol 24: 657-79
110. Dubois S, Mariner J, Waldmann TA, Tagaya Y. 2002. IL-15Ralpha recycles and presents
IL-15 In trans to neighboring cells. Immunity 17: 537-47
111. Lucas M, Schachterle W, Oberle K, Aichele P, Diefenbach A. 2007. Dendritic cells
prime natural killer cells by trans-presenting interleukin 15. Immunity 26: 503-17
112. Mortier E, Woo T, Advincula R, Gozalo S, Ma A. 2008. IL-15Ralpha chaperones IL-15
to stable dendritic cell membrane complexes that activate NK cells via trans presentation.
J Exp Med 205: 1213-25
113. Cooper MA, Bush JE, Fehniger TA, VanDeusen JB, Waite RE, Liu Y, Aguila HL,
Caligiuri MA. 2002. In vivo evidence for a dependence on interleukin 15 for survival of
natural killer cells. Blood 100: 3633-8
114. Huntington ND, Puthalakath H, Gunn P, Naik E, Michalak EM, Smyth MJ, Tabarias H,
Degli-Esposti MA, Dewson G, Willis SN, Motoyama N, Huang DC, Nutt SL, Tarlinton
DM, Strasser A. 2007. Interleukin 15-mediated survival of natural killer cells is
determined by interactions among Bim, Noxa and Mcl-1. Nat Immunol 8: 856-63
115. Ranson T, Vosshenrich CA, Corcuff E, Richard O, Muller W, Di Santo JP. 2003. IL-15 is
an essential mediator of peripheral NK-cell homeostasis. Blood 101: 4887-93
116. Gregoire C, Chasson L, Luci C, Tomasello E, Geissmann F, Vivier E, Walzer T. 2007.
The trafficking of natural killer cells. Immunol Rev 220: 169-82
117. Jamieson AM, Isnard P, Dorfman JR, Coles MC, Raulet DH. 2004. Turnover and
proliferation of NK cells in steady state and lymphopenic conditions. J Immunol 172:
864-70
121
118. Walzer T, Blery M, Chaix J, Fuseri N, Chasson L, Robbins SH, Jaeger S, Andre P,
Gauthier L, Daniel L, Chemin K, Morel Y, Dalod M, Imbert J, Pierres M, Moretta A,
Romagne F, Vivier E. 2007. Identification, activation, and selective in vivo ablation of
mouse NK cells via NKp46. Proc Natl Acad Sci U S A 104: 3384-9
119. Zhang Y, Wallace DL, de Lara CM, Ghattas H, Asquith B, Worth A, Griffin GE, Taylor
GP, Tough DF, Beverley PC, Macallan DC. 2007. In vivo kinetics of human natural
killer cells: the effects of ageing and acute and chronic viral infection. Immunology 121:
258-65
120. Prlic M, Blazar BR, Farrar MA, Jameson SC. 2003. In vivo survival and homeostatic
proliferation of natural killer cells. J Exp Med 197: 967-76
121. Sun JC, Beilke JN, Bezman NA, Lanier LL. 2011. Homeostatic proliferation generates
long-lived natural killer cells that respond against viral infection. J Exp Med 208: 357-68
122. Chiossone L, Chaix J, Fuseri N, Roth C, Vivier E, Walzer T. 2009. Maturation of mouse
NK cells is a 4-stage developmental program. Blood 113: 5488-96
123. Hayakawa Y, Smyth MJ. 2006. CD27 dissects mature NK cells into two subsets with
distinct responsiveness and migratory capacity. J Immunol 176: 1517-24
124. Cooper MA, Fehniger TA, Caligiuri MA. 2001. The biology of human natural killer-cell
subsets. Trends Immunol 22: 633-40
125. Raulet DH, Vance RE. 2006. Self-tolerance of natural killer cells. Nat Rev Immunol 6:
520-31
126. Lanier LL. 2005. NK cell recognition. Annu Rev Immunol 23: 225-74
127. Diefenbach A, Tomasello E, Lucas M, Jamieson AM, Hsia JK, Vivier E, Raulet DH.
2002. Selective associations with signaling proteins determine stimulatory versus
costimulatory activity of NKG2D. Nat Immunol 3: 1142-9
128. Gilfillan S, Ho EL, Cella M, Yokoyama WM, Colonna M. 2002. NKG2D recruits two
distinct adapters to trigger NK cell activation and costimulation. Nat Immunol 3: 1150-5
129. Billadeau DD, Upshaw JL, Schoon RA, Dick CJ, Leibson PJ. 2003. NKG2D-DAP10
triggers human NK cell-mediated killing via a Syk-independent regulatory pathway. Nat
Immunol 4: 557-64
130. Jost S, Altfeld M. 2013. Control of human viral infections by natural killer cells. Annu
Rev Immunol 31: 163-94
131. Lanier LL, Corliss BC, Wu J, Leong C, Phillips JH. 1998. Immunoreceptor DAP12
bearing a tyrosine-based activation motif is involved in activating NK cells. Nature 391:
703-7
132. Wu J, Song Y, Bakker AB, Bauer S, Spies T, Lanier LL, Phillips JH. 1999. An activating
immunoreceptor complex formed by NKG2D and DAP10. Science 285: 730-2
133. Raulet DH, Gasser S, Gowen BG, Deng W, Jung H. 2013. Regulation of ligands for the
NKG2D activating receptor. Annu Rev Immunol 31: 413-41
134. Carayannopoulos LN, Naidenko OV, Fremont DH, Yokoyama WM. 2002. Cutting edge:
murine UL16-binding protein-like transcript 1: a newly described transcript encoding a
high-affinity ligand for murine NKG2D. J Immunol 169: 4079-83
135. Diefenbach A, Hsia JK, Hsiung MY, Raulet DH. 2003. A novel ligand for the NKG2D
receptor activates NK cells and macrophages and induces tumor immunity. Eur J
Immunol 33: 381-91
136. Nice TJ, Coscoy L, Raulet DH. 2009. Posttranslational regulation of the NKG2D ligand
Mult1 in response to cell stress. J Exp Med 206: 287-98
122
137. Nice TJ, Deng W, Coscoy L, Raulet DH. 2010. Stress-regulated targeting of the NKG2D
ligand Mult1 by a membrane-associated RING-CH family E3 ligase. J Immunol 185:
5369-76
138. Yokoyama WM, Riley JK. 2008. NK cells and their receptors. Reprod Biomed Online 16:
173-91
139. Mandelboim O, Lieberman N, Lev M, Paul L, Arnon TI, Bushkin Y, Davis DM,
Strominger JL, Yewdell JW, Porgador A. 2001. Recognition of haemagglutinins on
virus-infected cells by NKp46 activates lysis by human NK cells. Nature 409: 1055-60
140. Bloushtain N, Qimron U, Bar-Ilan A, Hershkovitz O, Gazit R, Fima E, Korc M,
Vlodavsky I, Bovin NV, Porgador A. 2004. Membrane-associated heparan sulfate
proteoglycans are involved in the recognition of cellular targets by NKp30 and NKp46. J
Immunol 173: 2392-401
141. Pogge von Strandmann E, Simhadri VR, von Tresckow B, Sasse S, Reiners KS, Hansen
HP, Rothe A, Boll B, Simhadri VL, Borchmann P, McKinnon PJ, Hallek M, Engert A.
2007. Human leukocyte antigen-B-associated transcript 3 is released from tumor cells
and engages the NKp30 receptor on natural killer cells. Immunity 27: 965-74
142. Halfteck GG, Elboim M, Gur C, Achdout H, Ghadially H, Mandelboim O. 2009.
Enhanced in vivo growth of lymphoma tumors in the absence of the NK-activating
receptor NKp46/NCR1. J Immunol 182: 2221-30
143. Pessino A, Sivori S, Bottino C, Malaspina A, Morelli L, Moretta L, Biassoni R, Moretta
A. 1998. Molecular cloning of NKp46: a novel member of the immunoglobulin
superfamily involved in triggering of natural cytotoxicity. J Exp Med 188: 953-60
144. Karre K, Ljunggren HG, Piontek G, Kiessling R. 1986. Selective rejection of H-2-
deficient lymphoma variants suggests alternative immune defence strategy. Nature 319:
675-8
145. Ljunggren HG, Karre K. 1990. In search of the 'missing self': MHC molecules and NK
cell recognition. Immunol Today 11: 237-44
146. Correa I, Raulet DH. 1995. Binding of diverse peptides to MHC class I molecules
inhibits target cell lysis by activated natural killer cells. Immunity 2: 61-71
147. Hanke T, Takizawa H, McMahon CW, Busch DH, Pamer EG, Miller JD, Altman JD, Liu
Y, Cado D, Lemonnier FA, Bjorkman PJ, Raulet DH. 1999. Direct assessment of MHC
class I binding by seven Ly49 inhibitory NK cell receptors. Immunity 11: 67-77
148. Orihuela M, Margulies DH, Yokoyama WM. 1996. The natural killer cell receptor Ly-
49A recognizes a peptide-induced conformational determinant on its major
histocompatibility complex class I ligand. Proc Natl Acad Sci U S A 93: 11792-7
149. Veinotte LL, Wilhelm BT, Mager DL, Takei F. 2003. Acquisition of MHC-specific
receptors on murine natural killer cells. Crit Rev Immunol 23: 251-66
150. Raulet DH, Held W, Correa I, Dorfman JR, Wu MF, Corral L. 1997. Specificity,
tolerance and developmental regulation of natural killer cells defined by expression of
class I-specific Ly49 receptors. Immunol Rev 155: 41-52
151. Doucey MA, Scarpellino L, Zimmer J, Guillaume P, Luescher IF, Bron C, Held W. 2004.
Cis association of Ly49A with MHC class I restricts natural killer cell inhibition. Nat
Immunol 5: 328-36
152. Scarpellino L, Oeschger F, Guillaume P, Coudert JD, Levy F, Leclercq G, Held W. 2007.
Interactions of Ly49 family receptors with MHC class I ligands in trans and cis. J
Immunol 178: 1277-84
123
153. Arase H, Mocarski ES, Campbell AE, Hill AB, Lanier LL. 2002. Direct recognition of
cytomegalovirus by activating and inhibitory NK cell receptors. Science 296: 1323-6
154. Smith HR, Heusel JW, Mehta IK, Kim S, Dorner BG, Naidenko OV, Iizuka K, Furukawa
H, Beckman DL, Pingel JT, Scalzo AA, Fremont DH, Yokoyama WM. 2002.
Recognition of a virus-encoded ligand by a natural killer cell activation receptor. Proc
Natl Acad Sci U S A 99: 8826-31
155. Barten R, Torkar M, Haude A, Trowsdale J, Wilson MJ. 2001. Divergent and convergent
evolution of NK-cell receptors. Trends Immunol 22: 52-7
156. Colonna M, Samaridis J. 1995. Cloning of immunoglobulin-superfamily members
associated with HLA-C and HLA-B recognition by human natural killer cells. Science
268: 405-8
157. Wagtmann N, Biassoni R, Cantoni C, Verdiani S, Malnati MS, Vitale M, Bottino C,
Moretta L, Moretta A, Long EO. 1995. Molecular clones of the p58 NK cell receptor
reveal immunoglobulin-related molecules with diversity in both the extra- and
intracellular domains. Immunity 2: 439-49
158. Pegram HJ, Andrews DM, Smyth MJ, Darcy PK, Kershaw MH. 2011. Activating and
inhibitory receptors of natural killer cells. Immunol Cell Biol 89: 216-24
159. Held W, Roland J, Raulet DH. 1995. Allelic exclusion of Ly49-family genes encoding
class I MHC-specific receptors on NK cells. Nature 376: 355-8
160. Held W, Raulet DH. 1997. Expression of the Ly49A gene in murine natural killer cell
clones is predominantly but not exclusively mono-allelic. Eur J Immunol 27: 2876-84
161. Saleh A, Davies GE, Pascal V, Wright PW, Hodge DL, Cho EH, Lockett SJ, Abshari M,
Anderson SK. 2004. Identification of probabilistic transcriptional switches in the Ly49
gene cluster: a eukaryotic mechanism for selective gene activation. Immunity 21: 55-66
162. Borrego F, Ulbrecht M, Weiss EH, Coligan JE, Brooks AG. 1998. Recognition of human
histocompatibility leukocyte antigen (HLA)-E complexed with HLA class I signal
sequence-derived peptides by CD94/NKG2 confers protection from natural killer cell-
mediated lysis. J Exp Med 187: 813-8
163. Lee N, Goodlett DR, Ishitani A, Marquardt H, Geraghty DE. 1998. HLA-E surface
expression depends on binding of TAP-dependent peptides derived from certain HLA
class I signal sequences. J Immunol 160: 4951-60
164. Mingari MC, Ponte M, Bertone S, Schiavetti F, Vitale C, Bellomo R, Moretta A, Moretta
L. 1998. HLA class I-specific inhibitory receptors in human T lymphocytes: interleukin
15-induced expression of CD94/NKG2A in superantigen- or alloantigen-activated CD8+
T cells. Proc Natl Acad Sci U S A 95: 1172-7
165. Kirkham CL, Carlyle JR. 2014. Complexity and Diversity of the NKR-P1:Clr
(Klrb1:Clec2) Recognition Systems. Front Immunol 5: 214
166. Aldemir H, Prod'homme V, Dumaurier MJ, Retiere C, Poupon G, Cazareth J, Bihl F,
Braud VM. 2005. Cutting edge: lectin-like transcript 1 is a ligand for the CD161 receptor.
J Immunol 175: 7791-5
167. Rosen DB, Bettadapura J, Alsharifi M, Mathew PA, Warren HS, Lanier LL. 2005.
Cutting edge: lectin-like transcript-1 is a ligand for the inhibitory human NKR-P1A
receptor. J Immunol 175: 7796-9
168. Tassi I, Colonna M. 2005. The cytotoxicity receptor CRACC (CS-1) recruits EAT-2 and
activates the PI3K and phospholipase Cgamma signaling pathways in human NK cells. J
Immunol 175: 7996-8002
124
169. Veillette A. 2006. NK cell regulation by SLAM family receptors and SAP-related
adapters. Immunol Rev 214: 22-34
170. Degli-Esposti MA, Smyth MJ. 2005. Close encounters of different kinds: dendritic cells
and NK cells take centre stage. Nat Rev Immunol 5: 112-24
171. Moretta L, Ferlazzo G, Bottino C, Vitale M, Pende D, Mingari MC, Moretta A. 2006.
Effector and regulatory events during natural killer-dendritic cell interactions. Immunol
Rev 214: 219-28
172. Walzer T, Dalod M, Robbins SH, Zitvogel L, Vivier E. 2005. Natural-killer cells and
dendritic cells: "l'union fait la force". Blood 106: 2252-8
173. Piccioli D, Sbrana S, Melandri E, Valiante NM. 2002. Contact-dependent stimulation and
inhibition of dendritic cells by natural killer cells. J Exp Med 195: 335-41
174. Martin-Fontecha A, Thomsen LL, Brett S, Gerard C, Lipp M, Lanzavecchia A, Sallusto
F. 2004. Induced recruitment of NK cells to lymph nodes provides IFN-gamma for T(H)1
priming. Nat Immunol 5: 1260-5
175. Morandi B, Bougras G, Muller WA, Ferlazzo G, Munz C. 2006. NK cells of human
secondary lymphoid tissues enhance T cell polarization via IFN-gamma secretion. Eur J
Immunol 36: 2394-400
176. Nakayama M, Takeda K, Kawano M, Takai T, Ishii N, Ogasawara K. 2011. Natural killer
(NK)-dendritic cell interactions generate MHC class II-dressed NK cells that regulate
CD4+ T cells. Proc Natl Acad Sci U S A 108: 18360-5
177. Takeda K, Dennert G. 1993. The development of autoimmunity in C57BL/6 lpr mice
correlates with the disappearance of natural killer type 1-positive cells: evidence for their
suppressive action on bone marrow stem cell proliferation, B cell immunoglobulin
secretion, and autoimmune symptoms. J Exp Med 177: 155-64
178. van Dommelen SL, Sumaria N, Schreiber RD, Scalzo AA, Smyth MJ, Degli-Esposti MA.
2006. Perforin and granzymes have distinct roles in defensive immunity and
immunopathology. Immunity 25: 835-48
179. Sun JC, Beilke JN, Lanier LL. 2009. Adaptive immune features of natural killer cells.
Nature 457: 557-61
180. Welsh RM, Waggoner SN. 2013. NK cells controlling virus-specific T cells: Rheostats
for acute vs. persistent infections. Virology 435: 37-45
181. Burshtyn DN. 2013. NK cells and poxvirus infection. Front Immunol 4: 7
182. Lee SH, Miyagi T, Biron CA. 2007. Keeping NK cells in highly regulated antiviral
warfare. Trends Immunol 28: 252-9
183. Scalzo AA, Corbett AJ, Rawlinson WD, Scott GM, Degli-Esposti MA. 2007. The
interplay between host and viral factors in shaping the outcome of cytomegalovirus
infection. Immunol Cell Biol 85: 46-54
184. Gazit R, Gruda R, Elboim M, Arnon TI, Katz G, Achdout H, Hanna J, Qimron U, Landau
G, Greenbaum E, Zakay-Rones Z, Porgador A, Mandelboim O. 2006. Lethal influenza
infection in the absence of the natural killer cell receptor gene Ncr1. Nat Immunol 7: 517-
23
185. Stein-Streilein J, Guffee J. 1986. In vivo treatment of mice and hamsters with antibodies
to asialo GM1 increases morbidity and mortality to pulmonary influenza infection. J
Immunol 136: 1435-41
186. Asmal M, Sun Y, Lane S, Yeh W, Schmidt SD, Mascola JR, Letvin NL. 2011. Antibody-
dependent cell-mediated viral inhibition emerges after simian immunodeficiency virus
125
SIVmac251 infection of rhesus monkeys coincident with gp140-binding antibodies and is
effective against neutralization-resistant viruses. J Virol 85: 5465-75
187. Hellmann I, Lim SY, Gelman RS, Letvin NL. 2011. Association of activating KIR copy
number variation of NK cells with containment of SIV replication in rhesus monkeys.
PLoS Pathog 7: e1002436
188. Sun Y, Asmal M, Lane S, Permar SR, Schmidt SD, Mascola JR, Letvin NL. 2011.
Antibody-dependent cell-mediated cytotoxicity in simian immunodeficiency virus-
infected rhesus monkeys. J Virol 85: 6906-12
189. Flores-Villanueva PO, Yunis EJ, Delgado JC, Vittinghoff E, Buchbinder S, Leung JY,
Uglialoro AM, Clavijo OP, Rosenberg ES, Kalams SA, Braun JD, Boswell SL, Walker
BD, Goldfeld AE. 2001. Control of HIV-1 viremia and protection from AIDS are
associated with HLA-Bw4 homozygosity. Proc Natl Acad Sci U S A 98: 5140-5
190. Martin MP, Gao X, Lee JH, Nelson GW, Detels R, Goedert JJ, Buchbinder S, Hoots K,
Vlahov D, Trowsdale J, Wilson M, O'Brien SJ, Carrington M. 2002. Epistatic interaction
between KIR3DS1 and HLA-B delays the progression to AIDS. Nat Genet 31: 429-34
191. Vidal SM, Lanier LL. 2006. NK cell recognition of mouse cytomegalovirus-infected
cells. Curr Top Microbiol Immunol 298: 183-206
192. Voigt S, Mesci A, Ettinger J, Fine JH, Chen P, Chou W, Carlyle JR. 2007.
Cytomegalovirus evasion of innate immunity by subversion of the NKR-P1B:Clr-b
missing-self axis. Immunity 26: 617-27
193. Williams KJ, Wilson E, Davidson CL, Aguilar OA, Fu L, Carlyle JR, Burshtyn DN.
2012. Poxvirus infection-associated downregulation of C-type lectin-related-b prevents
NK cell inhibition by NK receptor protein-1B. J Immunol 188: 4980-91
194. Fine JH, Chen P, Mesci A, Allan DS, Gasser S, Raulet DH, Carlyle JR. 2010.
Chemotherapy-induced genotoxic stress promotes sensitivity to natural killer cell
cytotoxicity by enabling missing-self recognition. Cancer Res 70: 7102-13
195. Norris BA, Uebelhoer LS, Nakaya HI, Price AA, Grakoui A, Pulendran B. 2013. Chronic
but not acute virus infection induces sustained expansion of myeloid suppressor cell
numbers that inhibit viral-specific T cell immunity. Immunity 38: 309-21
196. Waggoner SN, Daniels KA, Welsh RM. 2014. Therapeutic depletion of natural killer
cells controls persistent infection. J Virol 88: 1953-60
197. Cesta MF. 2006. Normal structure, function, and histology of the spleen. Toxicol Pathol
34: 455-65
198. Mebius RE, Kraal G. 2005. Structure and function of the spleen. Nat Rev Immunol 5:
606-16
199. Green MC. 1967. A defect of the splanchnic mesoderm caused by the mutant gene
dominant hemimelia in the mouse. Dev Biol 15: 62-89
200. Hecksher-Sorensen J, Watson RP, Lettice LA, Serup P, Eley L, De Angelis C, Ahlgren
U, Hill RE. 2004. The splanchnic mesodermal plate directs spleen and pancreatic
laterality, and is regulated by Bapx1/Nkx3.2. Development 131: 4665-75
201. Herzer U, Crocoll A, Barton D, Howells N, Englert C. 1999. The Wilms tumor
suppressor gene wt1 is required for development of the spleen. Curr Biol 9: 837-40
202. Lu J, Chang P, Richardson JA, Gan L, Weiler H, Olson EN. 2000. The basic helix-loop-
helix transcription factor capsulin controls spleen organogenesis. Proc Natl Acad Sci U S
A 97: 9525-30
126
203. Roberts CW, Shutter JR, Korsmeyer SJ. 1994. Hox11 controls the genesis of the spleen.
Nature 368: 747-9
204. Brendolan A, Ferretti E, Salsi V, Moses K, Quaggin S, Blasi F, Cleary ML, Selleri L.
2005. A Pbx1-dependent genetic and transcriptional network regulates spleen ontogeny.
Development 132: 3113-26
205. Mebius RE, Rennert P, Weissman IL. 1997. Developing lymph nodes collect CD4+CD3-
LTbeta+ cells that can differentiate to APC, NK cells, and follicular cells but not T or B
cells. Immunity 7: 493-504
206. Seifert MF, Marks SC, Jr. 1985. The regulation of hemopoiesis in the spleen. Experientia
41: 192-9
207. Ansel KM, Ngo VN, Hyman PL, Luther SA, Forster R, Sedgwick JD, Browning JL, Lipp
M, Cyster JG. 2000. A chemokine-driven positive feedback loop organizes lymphoid
follicles. Nature 406: 309-14
208. Dejardin E, Droin NM, Delhase M, Haas E, Cao Y, Makris C, Li ZW, Karin M, Ware
CF, Green DR. 2002. The lymphotoxin-beta receptor induces different patterns of gene
expression via two NF-kappaB pathways. Immunity 17: 525-35
209. Vondenhoff MF, Desanti GE, Cupedo T, Bertrand JY, Cumano A, Kraal G, Mebius RE,
Golub R. 2008. Separation of splenic red and white pulp occurs before birth in a
LTalphabeta-independent manner. J Leukoc Biol 84: 152-61
210. Katakai T, Suto H, Sugai M, Gonda H, Togawa A, Suematsu S, Ebisuno Y, Katagiri K,
Kinashi T, Shimizu A. 2008. Organizer-like reticular stromal cell layer common to adult
secondary lymphoid organs. J Immunol 181: 6189-200
211. Koning JJ, Mebius RE. 2012. Interdependence of stromal and immune cells for lymph
node function. Trends Immunol 33: 264-70
212. Mueller SN, Germain RN. 2009. Stromal cell contributions to the homeostasis and
functionality of the immune system. Nat Rev Immunol 9: 618-29
213. Castagnaro L, Lenti E, Maruzzelli S, Spinardi L, Migliori E, Farinello D, Sitia G,
Harrelson Z, Evans SM, Guidotti LG, Harvey RP, Brendolan A. 2013. Nkx2-
5(+)islet1(+) mesenchymal precursors generate distinct spleen stromal cell subsets and
participate in restoring stromal network integrity. Immunity 38: 782-91
214. Shinkura R, Kitada K, Matsuda F, Tashiro K, Ikuta K, Suzuki M, Kogishi K, Serikawa T,
Honjo T. 1999. Alymphoplasia is caused by a point mutation in the mouse gene encoding
Nf-kappa b-inducing kinase. Nat Genet 22: 74-7
215. De Togni P, Goellner J, Ruddle NH, Streeter PR, Fick A, Mariathasan S, Smith SC,
Carlson R, Shornick LP, Strauss-Schoenberger J, et al. 1994. Abnormal development of
peripheral lymphoid organs in mice deficient in lymphotoxin. Science 264: 703-7
216. Miyawaki S, Nakamura Y, Suzuka H, Koba M, Yasumizu R, Ikehara S, Shibata Y. 1994.
A new mutation, aly, that induces a generalized lack of lymph nodes accompanied by
immunodeficiency in mice. Eur J Immunol 24: 429-34
217. Bronte V, Pittet MJ. 2013. The spleen in local and systemic regulation of immunity.
Immunity 39: 806-18
218. Luther SA, Tang HL, Hyman PL, Farr AG, Cyster JG. 2000. Coexpression of the
chemokines ELC and SLC by T zone stromal cells and deletion of the ELC gene in the
plt/plt mouse. Proc Natl Acad Sci U S A 97: 12694-9
219. Ngo VN, Korner H, Gunn MD, Schmidt KN, Riminton DS, Cooper MD, Browning JL,
Sedgwick JD, Cyster JG. 1999. Lymphotoxin alpha/beta and tumor necrosis factor are
127
required for stromal cell expression of homing chemokines in B and T cell areas of the
spleen. J Exp Med 189: 403-12
220. Turley SJ, Fletcher AL, Elpek KG. 2010. The stromal and haematopoietic antigen-
presenting cells that reside in secondary lymphoid organs. Nat Rev Immunol 10: 813-25
221. Karrer U, Althage A, Odermatt B, Roberts CW, Korsmeyer SJ, Miyawaki S, Hengartner
H, Zinkernagel RM. 1997. On the key role of secondary lymphoid organs in antiviral
immune responses studied in alymphoplastic (aly/aly) and spleenless (Hox11(-)/-) mutant
mice. J Exp Med 185: 2157-70
222. Ciavarra RP, Buhrer K, Van Rooijen N, Tedeschi B. 1997. T cell priming against
vesicular stomatitis virus analyzed in situ: red pulp macrophages, but neither marginal
metallophilic nor marginal zone macrophages, are required for priming CD4+ and CD8+
T cells. J Immunol 158: 1749-55
223. Seiler P, Aichele P, Odermatt B, Hengartner H, Zinkernagel RM, Schwendener RA.
1997. Crucial role of marginal zone macrophages and marginal zone metallophils in the
clearance of lymphocytic choriomeningitis virus infection. Eur J Immunol 27: 2626-33
224. Al-Salem AH. 2011. Splenic complications of sickle cell anemia and the role of
splenectomy. ISRN Hematol 2011: 864257
225. Lau FY. 1959. Pulmonary infarction and atrophy of the spleen associated with sickle-cell
hemoglobin C disease. N Engl J Med 260: 907-11
226. Wardrop CA, Dagg JH, Lee FD, Singh H, Dyet JF, Moffat A. 1975. Immunological
abnormalities in splenic atrophy. Lancet 2: 4-7
227. Coppo P, Saadoun D, Varet B. 2006. Autoimmune manifestations in acquired idiopathic
splenic atrophy: A puzzling association. Eur J Intern Med 17: 580-2
228. Di Sabatino A, Brunetti L, Carnevale Maffe G, Giuffrida P, Corazza GR. 2013. Is it
worth investigating splenic function in patients with celiac disease? World J
Gastroenterol 19: 2313-8
229. Di Sabatino A, Rosado MM, Cazzola P, Riboni R, Biagi F, Carsetti R, Corazza GR.
2006. Splenic hypofunction and the spectrum of autoimmune and malignant
complications in celiac disease. Clin Gastroenterol Hepatol 4: 179-86
230. Harmon GS, Lee JS. 2010. Splenic atrophy in celiac disease. Clin Gastroenterol Hepatol
8: A22
231. Trewby PN, Chipping PM, Palmer SJ, Roberts PD, Lewis SM, Stewart JS. 1981. Splenic
atrophy in adult coeliac disease: is it reversible? Gut 22: 628-32
232. Santilli D, Govoni M, Prandini N, Rizzo N, Trotta F. 2003. Autosplenectomy and
antiphospholipid antibodies in systemic lupus erythematosus: A pathogenetic
relationship? Semin Arthritis Rheum 33: 125-33
233. Santos N, Silva R, Rodrigues J, Torres-Costa J. 2014. Sjogren's syndrome and acquired
splenic atrophy with septic shock: a case report. J Med Case Rep 8: 10
234. Zhan J, Deng R, Tang J, Zhang B, Tang Y, Wang JK, Li F, Anderson VM, McNutt MA,
Gu J. 2006. The spleen as a target in severe acute respiratory syndrome. FASEB J 20:
2321-8
235. To KF, Chan PK, Chan KF, Lee WK, Lam WY, Wong KF, Tang NL, Tsang DN, Sung
RY, Buckley TA, Tam JS, Cheng AF. 2001. Pathology of fatal human infection
associated with avian influenza A H5N1 virus. J Med Virol 63: 242-6
128
236. Matteucci D, Toniolo A, Conaldi PG, Basolo F, Gori Z, Bendinelli M. 1985. Systemic
lymphoid atrophy in coxsackievirus B3-infected mice: effects of virus and
immunopotentiating agents. J Infect Dis 151: 1100-8
237. Shibuta H, Adachi A, Kanda T, Matumoto M. 1982. Experimental parainfluenzavirus
infection in mice: fatal illness with atrophy of thymus and spleen in mice caused by a
variant of parainfluenza 3 virus. Infect Immun 35: 437-41
238. Bartholdy C, Hogh-Petersen M, Storm P, Holst PJ, Orskov C, Christensen JP, Thomsen
AR. 2014. IFNgamma and perforin cooperate to control infection and prevent fatal
pathology during persistent gammaherpesvirus infection in mice. Scand J Immunol 79:
395-403
239. Dutia BM, Clarke CJ, Allen DJ, Nash AA. 1997. Pathological changes in the spleens of
gamma interferon receptor-deficient mice infected with murine gammaherpesvirus: a role
for CD8 T cells. J Virol 71: 4278-83
240. Hayasaka D, Nagata N, Fujii Y, Hasegawa H, Sata T, Suzuki R, Gould EA, Takashima I,
Koike S. 2009. Mortality following peripheral infection with tick-borne encephalitis virus
results from a combination of central nervous system pathology, systemic inflammatory
and stress responses. Virology 390: 139-50
241. Di Sabatino A, Corazza GR. 2009. Coeliac disease. Lancet 373: 1480-93
242. Lewis NR, Holmes GK. 2010. Risk of morbidity in contemporary celiac disease. Expert
Rev Gastroenterol Hepatol 4: 767-80
243. Morelli AE, Larregina AT, Shufesky WJ, Zahorchak AF, Logar AJ, Papworth GD, Wang
Z, Watkins SC, Falo LD, Jr., Thomson AW. 2003. Internalization of circulating apoptotic
cells by splenic marginal zone dendritic cells: dependence on complement receptors and
effect on cytokine production. Blood 101: 611-20
244. Rathmell JC, Townsend SE, Xu JC, Flavell RA, Goodnow CC. 1996. Expansion or
elimination of B cells in vivo: dual roles for CD40- and Fas (CD95)-ligands modulated
by the B cell antigen receptor. Cell 87: 319-29
245. Steinman RM, Pack M, Inaba K. 1997. Dendritic cells in the T-cell areas of lymphoid
organs. Immunol Rev 156: 25-37
246. Offner H, Subramanian S, Parker SM, Wang C, Afentoulis ME, Lewis A, Vandenbark
AA, Hurn PD. 2006. Splenic atrophy in experimental stroke is accompanied by increased
regulatory T cells and circulating macrophages. J Immunol 176: 6523-31
247. Savino W. 2006. The thymus is a common target organ in infectious diseases. PLoS
Pathog 2: e62
248. Di Sabatino A, Carsetti R, Corazza GR. 2011. Post-splenectomy and hyposplenic states.
Lancet 378: 86-97
249. Foster PN, Hardy GJ, Losowsky MS. 1984. Fatal Salmonella septicaemia in a patient
with systemic lupus erythematosus and splenic atrophy. Br J Clin Pract 38: 434-5
250. Logan RF, Rifkind EA, Turner ID, Ferguson A. 1989. Mortality in celiac disease.
Gastroenterology 97: 265-71
251. Holdsworth RJ. 1991. Regeneration of the spleen and splenic autotransplantation. Br J
Surg 78: 270-8
252. Fenner F. 1976. Classification and nomenclature of viruses. Second report of the
International Committee on Taxonomy of Viruses. Intervirology 7: 1-115
129
253. Perez M, Craven RC, de la Torre JC. 2003. The small RING finger protein Z drives
arenavirus budding: implications for antiviral strategies. Proc Natl Acad Sci U S A 100:
12978-83
254. Lee KJ, Novella IS, Teng MN, Oldstone MB, de La Torre JC. 2000. NP and L proteins of
lymphocytic choriomeningitis virus (LCMV) are sufficient for efficient transcription and
replication of LCMV genomic RNA analogs. J Virol 74: 3470-7
255. Borrow P, Oldstone MB. 1992. Characterization of lymphocytic choriomeningitis virus-
binding protein(s): a candidate cellular receptor for the virus. J Virol 66: 7270-81
256. Parekh BS, Buchmeier MJ. 1986. Proteins of lymphocytic choriomeningitis virus:
antigenic topography of the viral glycoproteins. Virology 153: 168-78
257. Cao W, Henry MD, Borrow P, Yamada H, Elder JH, Ravkov EV, Nichol ST, Compans
RW, Campbell KP, Oldstone MB. 1998. Identification of alpha-dystroglycan as a
receptor for lymphocytic choriomeningitis virus and Lassa fever virus. Science 282:
2079-81
258. Ahmed R, Salmi A, Butler LD, Chiller JM, Oldstone MB. 1984. Selection of genetic
variants of lymphocytic choriomeningitis virus in spleens of persistently infected mice.
Role in suppression of cytotoxic T lymphocyte response and viral persistence. J Exp Med
160: 521-40
259. Zinkernagel RM, Doherty PC. 1979. MHC-restricted cytotoxic T cells: studies on the
biological role of polymorphic major transplantation antigens determining T-cell
restriction-specificity, function, and responsiveness. Adv Immunol 27: 51-177
260. Pircher H, Burki K, Lang R, Hengartner H, Zinkernagel RM. 1989. Tolerance induction
in double specific T-cell receptor transgenic mice varies with antigen. Nature 342: 559-
61
261. Tishon A, Borrow P, Evans C, Oldstone MB. 1993. Virus-induced immunosuppression.
1. Age at infection relates to a selective or generalized defect. Virology 195: 397-405
262. von Herrath MG, Guerder S, Lewicki H, Flavell RA, Oldstone MB. 1995. Coexpression
of B7-1 and viral ("self") transgenes in pancreatic beta cells can break peripheral
ignorance and lead to spontaneous autoimmune diabetes. Immunity 3: 727-38
263. Borrow P, Lewicki H, Wei X, Horwitz MS, Peffer N, Meyers H, Nelson JA, Gairin JE,
Hahn BH, Oldstone MB, Shaw GM. 1997. Antiviral pressure exerted by HIV-1-specific
cytotoxic T lymphocytes (CTLs) during primary infection demonstrated by rapid
selection of CTL escape virus. Nat Med 3: 205-11
264. Ahmed R, Hahn CS, Somasundaram T, Villarete L, Matloubian M, Strauss JH. 1991.
Molecular basis of organ-specific selection of viral variants during chronic infection. J
Virol 65: 4242-7
265. Matloubian M, Kolhekar SR, Somasundaram T, Ahmed R. 1993. Molecular determinants
of macrophage tropism and viral persistence: importance of single amino acid changes in
the polymerase and glycoprotein of lymphocytic choriomeningitis virus. J Virol 67:
7340-9
266. Wherry EJ, Blattman JN, Murali-Krishna K, van der Most R, Ahmed R. 2003. Viral
persistence alters CD8 T-cell immunodominance and tissue distribution and results in
distinct stages of functional impairment. J Virol 77: 4911-27
267. Sullivan BM, Emonet SF, Welch MJ, Lee AM, Campbell KP, de la Torre JC, Oldstone
MB. 2011. Point mutation in the glycoprotein of lymphocytic choriomeningitis virus is
130
necessary for receptor binding, dendritic cell infection, and long-term persistence. Proc
Natl Acad Sci U S A 108: 2969-74
268. Matloubian M, Somasundaram T, Kolhekar SR, Selvakumar R, Ahmed R. 1990. Genetic
basis of viral persistence: single amino acid change in the viral glycoprotein affects
ability of lymphocytic choriomeningitis virus to persist in adult mice. J Exp Med 172:
1043-8
269. Salvato M, Borrow P, Shimomaye E, Oldstone MB. 1991. Molecular basis of viral
persistence: a single amino acid change in the glycoprotein of lymphocytic
choriomeningitis virus is associated with suppression of the antiviral cytotoxic T-
lymphocyte response and establishment of persistence. J Virol 65: 1863-9
270. Sevilla N, Kunz S, Holz A, Lewicki H, Homann D, Yamada H, Campbell KP, de La
Torre JC, Oldstone MB. 2000. Immunosuppression and resultant viral persistence by
specific viral targeting of dendritic cells. J Exp Med 192: 1249-60
271. Kunz S, Sevilla N, McGavern DB, Campbell KP, Oldstone MB. 2001. Molecular
analysis of the interaction of LCMV with its cellular receptor [alpha]-dystroglycan. J Cell
Biol 155: 301-10
272. Smelt SC, Borrow P, Kunz S, Cao W, Tishon A, Lewicki H, Campbell KP, Oldstone
MB. 2001. Differences in affinity of binding of lymphocytic choriomeningitis virus
strains to the cellular receptor alpha-dystroglycan correlate with viral tropism and disease
kinetics. J Virol 75: 448-57
273. Borrow P, Evans CF, Oldstone MB. 1995. Virus-induced immunosuppression: immune
system-mediated destruction of virus-infected dendritic cells results in generalized
immune suppression. J Virol 69: 1059-70
274. Hahm B, Trifilo MJ, Zuniga EI, Oldstone MB. 2005. Viruses evade the immune system
through type I interferon-mediated STAT2-dependent, but STAT1-independent,
signaling. Immunity 22: 247-57
275. Wilson EB, Kidani Y, Elsaesser H, Barnard J, Raff L, Karp CL, Bensinger S, Brooks
DG. 2012. Emergence of distinct multiarmed immunoregulatory antigen-presenting cells
during persistent viral infection. Cell Host Microbe 11: 481-91
276. Sevilla N, Kunz S, McGavern D, Oldstone MB. 2003. Infection of dendritic cells by
lymphocytic choriomeningitis virus. Curr Top Microbiol Immunol 276: 125-44
277. Kunz S, Sevilla N, Rojek JM, Oldstone MB. 2004. Use of alternative receptors different
than alpha-dystroglycan by selected isolates of lymphocytic choriomeningitis virus.
Virology 325: 432-45
278. Shimojima M, Kawaoka Y. 2012. Cell surface molecules involved in infection mediated
by lymphocytic choriomeningitis virus glycoprotein. J Vet Med Sci 74: 1363-6
279. Shimojima M, Stroher U, Ebihara H, Feldmann H, Kawaoka Y. 2012. Identification of
cell surface molecules involved in dystroglycan-independent Lassa virus cell entry. J
Virol 86: 2067-78
280. Sullivan BM, Welch MJ, Lemke G, Oldstone MB. 2013. Is the TAM receptor Axl a
receptor for lymphocytic choriomeningitis virus? J Virol 87: 4071-4
281. Bachmann MF, Barner M, Viola A, Kopf M. 1999. Distinct kinetics of cytokine
production and cytolysis in effector and memory T cells after viral infection. Eur J
Immunol 29: 291-9
282. Grayson JM, Murali-Krishna K, Altman JD, Ahmed R. 2001. Gene expression in
antigen-specific CD8+ T cells during viral infection. J Immunol 166: 795-9
131
283. Kaech SM, Ahmed R. 2001. Memory CD8+ T cell differentiation: initial antigen
encounter triggers a developmental program in naive cells. Nat Immunol 2: 415-22
284. Schulz M, Aichele P, Vollenweider M, Bobe FW, Cardinaux F, Hengartner H,
Zinkernagel RM. 1989. Major histocompatibility complex--dependent T cell epitopes of
lymphocytic choriomeningitis virus nucleoprotein and their protective capacity against
viral disease. Eur J Immunol 19: 1657-67
285. Klavinskis LS, Whitton JL, Joly E, Oldstone MB. 1990. Vaccination and protection from
a lethal viral infection: identification, incorporation, and use of a cytotoxic T lymphocyte
glycoprotein epitope. Virology 178: 393-400
286. Gairin JE, Mazarguil H, Hudrisier D, Oldstone MB. 1995. Optimal lymphocytic
choriomeningitis virus sequences restricted by H-2Db major histocompatibility complex
class I molecules and presented to cytotoxic T lymphocytes. J Virol 69: 2297-305
287. Oldstone MB, Lewicki H, Borrow P, Hudrisier D, Gairin JE. 1995. Discriminated
selection among viral peptides with the appropriate anchor residues: implications for the
size of the cytotoxic T-lymphocyte repertoire and control of viral infection. J Virol 69:
7423-9
288. Selin LK, Vergilis K, Welsh RM, Nahill SR. 1996. Reduction of otherwise remarkably
stable virus-specific cytotoxic T lymphocyte memory by heterologous viral infections. J
Exp Med 183: 2489-99
289. Butz EA, Bevan MJ. 1998. Massive expansion of antigen-specific CD8+ T cells during
an acute virus infection. Immunity 8: 167-75
290. Murali-Krishna K, Altman JD, Suresh M, Sourdive DJ, Zajac AJ, Miller JD, Slansky J,
Ahmed R. 1998. Counting antigen-specific CD8 T cells: a reevaluation of bystander
activation during viral infection. Immunity 8: 177-87
291. Wang XZ, Stepp SE, Brehm MA, Chen HD, Selin LK, Welsh RM. 2003. Virus-specific
CD8 T cells in peripheral tissues are more resistant to apoptosis than those in lymphoid
organs. Immunity 18: 631-42
292. Lau LL, Jamieson BD, Somasundaram T, Ahmed R. 1994. Cytotoxic T-cell memory
without antigen. Nature 369: 648-52
293. Asano MS, Ahmed R. 1996. CD8 T cell memory in B cell-deficient mice. J Exp Med
183: 2165-74
294. Gallimore A, Glithero A, Godkin A, Tissot AC, Pluckthun A, Elliott T, Hengartner H,
Zinkernagel R. 1998. Induction and exhaustion of lymphocytic choriomeningitis virus-
specific cytotoxic T lymphocytes visualized using soluble tetrameric major
histocompatibility complex class I-peptide complexes. J Exp Med 187: 1383-93
295. Zajac AJ, Blattman JN, Murali-Krishna K, Sourdive DJ, Suresh M, Altman JD, Ahmed
R. 1998. Viral immune evasion due to persistence of activated T cells without effector
function. J Exp Med 188: 2205-13
296. Kaech SM, Wherry EJ, Ahmed R. 2002. Effector and memory T-cell differentiation:
implications for vaccine development. Nat Rev Immunol 2: 251-62
297. Khanolkar A, Fuller MJ, Zajac AJ. 2002. T cell responses to viral infections: lessons
from lymphocytic choriomeningitis virus. Immunol Res 26: 309-21
298. Wherry EJ. 2011. T cell exhaustion. Nat Immunol 12: 492-9
299. Barber DL, Wherry EJ, Masopust D, Zhu B, Allison JP, Sharpe AH, Freeman GJ, Ahmed
R. 2006. Restoring function in exhausted CD8 T cells during chronic viral infection.
Nature 439: 682-7
132
300. Brooks DG, Trifilo MJ, Edelmann KH, Teyton L, McGavern DB, Oldstone MB. 2006.
Interleukin-10 determines viral clearance or persistence in vivo. Nat Med 12: 1301-9
301. Ejrnaes M, Filippi CM, Martinic MM, Ling EM, Togher LM, Crotty S, von Herrath MG.
2006. Resolution of a chronic viral infection after interleukin-10 receptor blockade. J Exp
Med 203: 2461-72
302. Ng CT, Oldstone MB. 2012. Infected CD8alpha- dendritic cells are the predominant
source of IL-10 during establishment of persistent viral infection. Proc Natl Acad Sci U S
A 109: 14116-21
303. Borrow P, Tishon A, Oldstone MB. 1991. Infection of lymphocytes by a virus that aborts
cytotoxic T lymphocyte activity and establishes persistent infection. J Exp Med 174: 203-
12
304. Foulds KE, Zenewicz LA, Shedlock DJ, Jiang J, Troy AE, Shen H. 2002. Cutting edge:
CD4 and CD8 T cells are intrinsically different in their proliferative responses. J
Immunol 168: 1528-32
305. Homann D, Teyton L, Oldstone MB. 2001. Differential regulation of antiviral T-cell
immunity results in stable CD8+ but declining CD4+ T-cell memory. Nat Med 7: 913-9
306. Varga SM, Welsh RM. 1998. Detection of a high frequency of virus-specific CD4+ T
cells during acute infection with lymphocytic choriomeningitis virus. J Immunol 161:
3215-8
307. Varga SM, Welsh RM. 2000. High frequency of virus-specific interleukin-2-producing
CD4(+) T cells and Th1 dominance during lymphocytic choriomeningitis virus infection.
J Virol 74: 4429-32
308. Sun JC, Williams MA, Bevan MJ. 2004. CD4+ T cells are required for the maintenance,
not programming, of memory CD8+ T cells after acute infection. Nat Immunol 5: 927-33
309. von Herrath MG, Yokoyama M, Dockter J, Oldstone MB, Whitton JL. 1996. CD4-
deficient mice have reduced levels of memory cytotoxic T lymphocytes after
immunization and show diminished resistance to subsequent virus challenge. J Virol 70:
1072-9
310. Brooks DG, Teyton L, Oldstone MB, McGavern DB. 2005. Intrinsic functional
dysregulation of CD4 T cells occurs rapidly following persistent viral infection. J Virol
79: 10514-27
311. Fuller MJ, Zajac AJ. 2003. Ablation of CD8 and CD4 T cell responses by high viral
loads. J Immunol 170: 477-86
312. Mothe BR, Stewart BS, Oseroff C, Bui HH, Stogiera S, Garcia Z, Dow C, Rodriguez-
Carreno MP, Kotturi M, Pasquetto V, Botten J, Crotty S, Janssen E, Buchmeier MJ, Sette
A. 2007. Chronic lymphocytic choriomeningitis virus infection actively down-regulates
CD4+ T cell responses directed against a broad range of epitopes. J Immunol 179: 1058-
67
313. Matloubian M, Concepcion RJ, Ahmed R. 1994. CD4+ T cells are required to sustain
CD8+ cytotoxic T-cell responses during chronic viral infection. J Virol 68: 8056-63
314. Elsaesser H, Sauer K, Brooks DG. 2009. IL-21 is required to control chronic viral
infection. Science 324: 1569-72
315. Frohlich A, Kisielow J, Schmitz I, Freigang S, Shamshiev AT, Weber J, Marsland BJ,
Oxenius A, Kopf M. 2009. IL-21R on T cells is critical for sustained functionality and
control of chronic viral infection. Science 324: 1576-80
133
316. Boettler T, Moeckel F, Cheng Y, Heeg M, Salek-Ardakani S, Crotty S, Croft M, von
Herrath MG. 2012. OX40 facilitates control of a persistent virus infection. PLoS Pathog
8: e1002913
317. Probst HC, van den Broek M. 2005. Priming of CTLs by lymphocytic choriomeningitis
virus depends on dendritic cells. J Immunol 174: 3920-4
318. Montoya M, Edwards MJ, Reid DM, Borrow P. 2005. Rapid activation of spleen
dendritic cell subsets following lymphocytic choriomeningitis virus infection of mice:
analysis of the involvement of type 1 IFN. J Immunol 174: 1851-61
319. Thompson LJ, Kolumam GA, Thomas S, Murali-Krishna K. 2006. Innate inflammatory
signals induced by various pathogens differentially dictate the IFN-I dependence of CD8
T cells for clonal expansion and memory formation. J Immunol 177: 1746-54
320. Wilson EB, Yamada DH, Elsaesser H, Herskovitz J, Deng J, Cheng G, Aronow BJ, Karp
CL, Brooks DG. 2013. Blockade of chronic type I interferon signaling to control
persistent LCMV infection. Science 340: 202-7
321. Mack EA, Kallal LE, Demers DA, Biron CA. 2011. Type 1 interferon induction of
natural killer cell gamma interferon production for defense during lymphocytic
choriomeningitis virus infection. MBio 2
322. Lang PA, Lang KS, Xu HC, Grusdat M, Parish IA, Recher M, Elford AR, Dhanji S,
Shaabani N, Tran CW, Dissanayake D, Rahbar R, Ghazarian M, Brustle A, Fine J, Chen
P, Weaver CT, Klose C, Diefenbach A, Haussinger D, Carlyle JR, Kaech SM, Mak TW,
Ohashi PS. 2012. Natural killer cell activation enhances immune pathology and promotes
chronic infection by limiting CD8+ T-cell immunity. Proc Natl Acad Sci U S A 109:
1210-5
323. Waggoner SN, Cornberg M, Selin LK, Welsh RM. 2012. Natural killer cells act as
rheostats modulating antiviral T cells. Nature 481: 394-8
324. Cook KD, Whitmire JK. 2013. The depletion of NK cells prevents T cell exhaustion to
efficiently control disseminating virus infection. J Immunol 190: 641-9
325. Waggoner SN, Taniguchi RT, Mathew PA, Kumar V, Welsh RM. 2010. Absence of
mouse 2B4 promotes NK cell-mediated killing of activated CD8+ T cells, leading to
prolonged viral persistence and altered pathogenesis. J Clin Invest 120: 1925-38
326. Crouse J, Bedenikovic G, Wiesel M, Ibberson M, Xenarios I, Von Laer D, Kalinke U,
Vivier E, Jonjic S, Oxenius A. 2014. Type I Interferons Protect T Cells against NK Cell
Attack Mediated by the Activating Receptor NCR1. Immunity 40: 961-73
327. Xu HC, Grusdat M, Pandyra AA, Polz R, Huang J, Sharma P, Deenen R, Kohrer K,
Rahbar R, Diefenbach A, Gibbert K, Lohning M, Hocker L, Waibler Z, Haussinger D,
Mak TW, Ohashi PS, Lang KS, Lang PA. 2014. Type I interferon protects antiviral
CD8(+) T cells from NK cell cytotoxicity. Immunity 40: 949-60
328. Andrews DM, Estcourt MJ, Andoniou CE, Wikstrom ME, Khong A, Voigt V, Fleming P,
Tabarias H, Hill GR, van der Most RG, Scalzo AA, Smyth MJ, Degli-Esposti MA. 2010.
Innate immunity defines the capacity of antiviral T cells to limit persistent infection. J
Exp Med 207: 1333-43
329. Su HC, Nguyen KB, Salazar-Mather TP, Ruzek MC, Dalod MY, Biron CA. 2001. NK
cell functions restrain T cell responses during viral infections. Eur J Immunol 31: 3048-
55
134
330. Littwitz E, Francois S, Dittmer U, Gibbert K. 2013. Distinct roles of NK cells in viral
immunity during different phases of acute Friend retrovirus infection. Retrovirology 10:
127
331. Zhou G, Juang SW, Kane KP. 2013. NK cells exacerbate the pathology of influenza virus
infection in mice. Eur J Immunol 43: 929-38
332. Khakoo SI, Thio CL, Martin MP, Brooks CR, Gao X, Astemborski J, Cheng J, Goedert
JJ, Vlahov D, Hilgartner M, Cox S, Little AM, Alexander GJ, Cramp ME, O'Brien SJ,
Rosenberg WM, Thomas DL, Carrington M. 2004. HLA and NK cell inhibitory receptor
genes in resolving hepatitis C virus infection. Science 305: 872-4
333. Knapp S, Warshow U, Hegazy D, Brackenbury L, Guha IN, Fowell A, Little AM,
Alexander GJ, Rosenberg WM, Cramp ME, Khakoo SI. 2010. Consistent beneficial
effects of killer cell immunoglobulin-like receptor 2DL3 and group 1 human leukocyte
antigen-C following exposure to hepatitis C virus. Hepatology 51: 1168-75
334. Paladino N, Flores AC, Marcos CY, Fainboim H, Theiler G, Arruvito L, Williams F,
Middleton D, Fainboim L. 2007. Increased frequencies of activating natural killer
receptors are associated with liver injury in individuals who do not eliminate hepatitis C
virus. Tissue Antigens 69 Suppl 1: 109-11
335. Alter G, Rihn S, Walter K, Nolting A, Martin M, Rosenberg ES, Miller JS, Carrington M,
Altfeld M. 2009. HLA class I subtype-dependent expansion of KIR3DS1+ and
KIR3DL1+ NK cells during acute human immunodeficiency virus type 1 infection. J
Virol 83: 6798-805
336. Jennes W, Verheyden S, Demanet C, Adje-Toure CA, Vuylsteke B, Nkengasong JN,
Kestens L. 2006. Cutting edge: resistance to HIV-1 infection among African female sex
workers is associated with inhibitory KIR in the absence of their HLA ligands. J Immunol
177: 6588-92
337. Martin MP, Qi Y, Gao X, Yamada E, Martin JN, Pereyra F, Colombo S, Brown EE,
Shupert WL, Phair J, Goedert JJ, Buchbinder S, Kirk GD, Telenti A, Connors M, O'Brien
SJ, Walker BD, Parham P, Deeks SG, McVicar DW, Carrington M. 2007. Innate
partnership of HLA-B and KIR3DL1 subtypes against HIV-1. Nat Genet 39: 733-40
338. Romero V, Azocar J, Zuniga J, Clavijo OP, Terreros D, Gu X, Husain Z, Chung RT,
Amos C, Yunis EJ. 2008. Interaction of NK inhibitory receptor genes with HLA-C and
MHC class II alleles in Hepatitis C virus infection outcome. Mol Immunol 45: 2429-36
339. Yawata M, Yawata N, Draghi M, Little AM, Partheniou F, Parham P. 2006. Roles for
HLA and KIR polymorphisms in natural killer cell repertoire selection and modulation of
effector function. J Exp Med 203: 633-45
340. Parham P. 2004. Immunology. NK cells lose their inhibition. Science 305: 786-7
341. Moesta AK, Norman PJ, Yawata M, Yawata N, Gleimer M, Parham P. 2008. Synergistic
polymorphism at two positions distal to the ligand-binding site makes KIR2DL2 a
stronger receptor for HLA-C than KIR2DL3. J Immunol 180: 3969-79
342. Koziel MJ. 2005. Cellular immune responses against hepatitis C virus. Clin Infect Dis 41
Suppl 1: S25-31
343. Alter G, Martin MP, Teigen N, Carr WH, Suscovich TJ, Schneidewind A, Streeck H,
Waring M, Meier A, Brander C, Lifson JD, Allen TM, Carrington M, Altfeld M. 2007.
Differential natural killer cell-mediated inhibition of HIV-1 replication based on distinct
KIR/HLA subtypes. J Exp Med 204: 3027-36
135
344. Biron CA, Nguyen KB, Pien GC. 2002. Innate immune responses to LCMV infections:
natural killer cells and cytokines. Curr Top Microbiol Immunol 263: 7-27
345. Louten J, van Rooijen N, Biron CA. 2006. Type 1 IFN deficiency in the absence of
normal splenic architecture during lymphocytic choriomeningitis virus infection. J
Immunol 177: 3266-72
346. Aichele P, Unsoeld H, Koschella M, Schweier O, Kalinke U, Vucikuja S. 2006. CD8 T
cells specific for lymphocytic choriomeningitis virus require type I IFN receptor for
clonal expansion. J Immunol 176: 4525-9
347. Biron CA, Sonnenfeld G, Welsh RM. 1984. Interferon induces natural killer cell
blastogenesis in vivo. J Leukoc Biol 35: 31-7
348. Gidlund M, Orn A, Wigzell H, Senik A, Gresser I. 1978. Enhanced NK cell activity in
mice injected with interferon and interferon inducers. Nature 273: 759-61
349. Kolumam GA, Thomas S, Thompson LJ, Sprent J, Murali-Krishna K. 2005. Type I
interferons act directly on CD8 T cells to allow clonal expansion and memory formation
in response to viral infection. J Exp Med 202: 637-50
350. Wiesel M, Kratky W, Oxenius A. 2011. Type I IFN substitutes for T cell help during
viral infections. J Immunol 186: 754-63
351. Ou R, Zhou S, Huang L, Moskophidis D. 2001. Critical role for alpha/beta and gamma
interferons in persistence of lymphocytic choriomeningitis virus by clonal exhaustion of
cytotoxic T cells. J Virol 75: 8407-23
352. Binder D, Fehr J, Hengartner H, Zinkernagel RM. 1997. Virus-induced transient bone
marrow aplasia: major role of interferon-alpha/beta during acute infection with the
noncytopathic lymphocytic choriomeningitis virus. J Exp Med 185: 517-30
353. Teijaro JR, Ng C, Lee AM, Sullivan BM, Sheehan KC, Welch M, Schreiber RD, de la
Torre JC, Oldstone MB. 2013. Persistent LCMV infection is controlled by blockade of
type I interferon signaling. Science 340: 207-11
354. Chen L, Borozan I, Feld J, Sun J, Tannis LL, Coltescu C, Heathcote J, Edwards AM,
McGilvray ID. 2005. Hepatic gene expression discriminates responders and
nonresponders in treatment of chronic hepatitis C viral infection. Gastroenterology 128:
1437-44
355. Sarasin-Filipowicz M, Oakeley EJ, Duong FH, Christen V, Terracciano L, Filipowicz W,
Heim MH. 2008. Interferon signaling and treatment outcome in chronic hepatitis C. Proc
Natl Acad Sci U S A 105: 7034-9
356. Guidotti LG, Chisari FV. 2006. Immunobiology and pathogenesis of viral hepatitis. Annu
Rev Pathol 1: 23-61
357. Su AI, Pezacki JP, Wodicka L, Brideau AD, Supekova L, Thimme R, Wieland S, Bukh J,
Purcell RH, Schultz PG, Chisari FV. 2002. Genomic analysis of the host response to
hepatitis C virus infection. Proc Natl Acad Sci U S A 99: 15669-74
358. Bosinger SE, Li Q, Gordon SN, Klatt NR, Duan L, Xu L, Francella N, Sidahmed A,
Smith AJ, Cramer EM, Zeng M, Masopust D, Carlis JV, Ran L, Vanderford TH,
Paiardini M, Isett RB, Baldwin DA, Else JG, Staprans SI, Silvestri G, Haase AT, Kelvin
DJ. 2009. Global genomic analysis reveals rapid control of a robust innate response in
SIV-infected sooty mangabeys. J Clin Invest 119: 3556-72
359. Jacquelin B, Mayau V, Targat B, Liovat AS, Kunkel D, Petitjean G, Dillies MA, Roques
P, Butor C, Silvestri G, Giavedoni LD, Lebon P, Barre-Sinoussi F, Benecke A, Muller-
136
Trutwin MC. 2009. Nonpathogenic SIV infection of African green monkeys induces a
strong but rapidly controlled type I IFN response. J Clin Invest 119: 3544-55
360. Sandler NG, Bosinger SE, Estes JD, Zhu RT, Tharp GK, Boritz E, Levin D,
Wijeyesinghe S, Makamdop KN, del Prete GQ, Hill BJ, Timmer JK, Reiss E, Yarden G,
Darko S, Contijoch E, Todd JP, Silvestri G, Nason M, Norgren RB, Jr., Keele BF, Rao S,
Langer JA, Lifson JD, Schreiber G, Douek DC. 2014. Type I interferon responses in
rhesus macaques prevent SIV infection and slow disease progression. Nature 511: 601-5
361. Sharpe J, Ahlgren U, Perry P, Hill B, Ross A, Hecksher-Sorensen J, Baldock R,
Davidson D. 2002. Optical projection tomography as a tool for 3D microscopy and gene
expression studies. Science 296: 541-5
362. Kumar V, Scandella E, Danuser R, Onder L, Nitschke M, Fukui Y, Halin C, Ludewig B,
Stein JV. 2010. Global lymphoid tissue remodeling during a viral infection is
orchestrated by a B cell-lymphotoxin-dependent pathway. Blood 115: 4725-33
363. Odermatt B, Eppler M, Leist TP, Hengartner H, Zinkernagel RM. 1991. Virus-triggered
acquired immunodeficiency by cytotoxic T-cell-dependent destruction of antigen-
presenting cells and lymph follicle structure. Proc Natl Acad Sci U S A 88: 8252-6
364. Matter M, Odermatt B, Yagita H, Nuoffer JM, Ochsenbein AF. 2006. Elimination of
chronic viral infection by blocking CD27 signaling. J Exp Med 203: 2145-55
365. Engwerda CR, Ato M, Cotterell SE, Mynott TL, Tschannerl A, Gorak-Stolinska PM,
Kaye PM. 2002. A role for tumor necrosis factor-alpha in remodeling the splenic
marginal zone during Leishmania donovani infection. Am J Pathol 161: 429-37
366. Mueller SN, Matloubian M, Clemens DM, Sharpe AH, Freeman GJ, Gangappa S, Larsen
CP, Ahmed R. 2007. Viral targeting of fibroblastic reticular cells contributes to
immunosuppression and persistence during chronic infection. Proc Natl Acad Sci U S A
104: 15430-5
367. Ng CT, Nayak BP, Schmedt C, Oldstone MB. 2012. Immortalized clones of fibroblastic
reticular cells activate virus-specific T cells during virus infection. Proc Natl Acad Sci U
S A 109: 7823-8
368. Mueller SN, Vanguri VK, Ha SJ, West EE, Keir ME, Glickman JN, Sharpe AH, Ahmed
R. 2010. PD-L1 has distinct functions in hematopoietic and nonhematopoietic cells in
regulating T cell responses during chronic infection in mice. J Clin Invest 120: 2508-15
369. Zeng M, Southern PJ, Reilly CS, Beilman GJ, Chipman JG, Schacker TW, Haase AT.
2012. Lymphoid tissue damage in HIV-1 infection depletes naive T cells and limits T cell
reconstitution after antiretroviral therapy. PLoS Pathog 8: e1002437
370. Scandella E, Bolinger B, Lattmann E, Miller S, Favre S, Littman DR, Finke D, Luther
SA, Junt T, Ludewig B. 2008. Restoration of lymphoid organ integrity through the
interaction of lymphoid tissue-inducer cells with stroma of the T cell zone. Nat Immunol
9: 667-75
371. Benedict CA, De Trez C, Schneider K, Ha S, Patterson G, Ware CF. 2006. Specific
remodeling of splenic architecture by cytomegalovirus. PLoS Pathog 2: e16
372. Alexander-Miller MA, Derby MA, Sarin A, Henkart PA, Berzofsky JA. 1998.
Supraoptimal peptide-major histocompatibility complex causes a decrease in bc1-2 levels
and allows tumor necrosis factor alpha receptor II-mediated apoptosis of cytotoxic T
lymphocytes. J Exp Med 188: 1391-9
373. Badovinac VP, Tvinnereim AR, Harty JT. 2000. Regulation of antigen-specific CD8+ T
cell homeostasis by perforin and interferon-gamma. Science 290: 1354-8
137
374. Opferman JT, Ober BT, Narayanan R, Ashton-Rickardt PG. 2001. Suicide induced by
cytolytic activity controls the differentiation of memory CD8(+) T lymphocytes. Int
Immunol 13: 411-9
375. Zheng L, Fisher G, Miller RE, Peschon J, Lynch DH, Lenardo MJ. 1995. Induction of
apoptosis in mature T cells by tumour necrosis factor. Nature 377: 348-51
376. Badovinac VP, Porter BB, Harty JT. 2002. Programmed contraction of CD8(+) T cells
after infection. Nat Immunol 3: 619-26
377. De Boer RJ, Homann D, Perelson AS. 2003. Different dynamics of CD4+ and CD8+ T
cell responses during and after acute lymphocytic choriomeningitis virus infection. J
Immunol 171: 3928-35
378. Kamperschroer C, Quinn DG. 1999. Quantification of epitope-specific MHC class-II-
restricted T cells following lymphocytic choriomeningitis virus infection. Cell Immunol
193: 134-46
379. Misumi I, Alirezaei M, Eam B, Su MA, Whitton JL, Whitmire JK. 2013. Differential T
cell responses to residual viral antigen prolong CD4+ T cell contraction following the
resolution of infection. J Immunol 191: 5655-68
380. Varga SM, Welsh RM. 1998. Stability of virus-specific CD4+ T cell frequencies from
acute infection into long term memory. J Immunol 161: 367-74
381. Williams MA, Ravkov EV, Bevan MJ. 2008. Rapid culling of the CD4+ T cell repertoire
in the transition from effector to memory. Immunity 28: 533-45
382. Nguyen LT, McKall-Faienza K, Zakarian A, Speiser DE, Mak TW, Ohashi PS. 2000.
TNF receptor 1 (TNFR1) and CD95 are not required for T cell deletion after virus
infection but contribute to peptide-induced deletion under limited conditions. Eur J
Immunol 30: 683-8
383. Reich A, Korner H, Sedgwick JD, Pircher H. 2000. Immune down-regulation and
peripheral deletion of CD8 T cells does not require TNF receptor-ligand interactions nor
CD95 (Fas, APO-1). Eur J Immunol 30: 678-82
384. Suresh M, Singh A, Fischer C. 2005. Role of tumor necrosis factor receptors in regulating
CD8 T-cell responses during acute lymphocytic choriomeningitis virus infection. J Virol
79: 202-13
385. Singh A, Suresh M. 2007. A role for TNF in limiting the duration of CTL effector phase
and magnitude of CD8 T cell memory. J Leukoc Biol 82: 1201-11
386. Suresh M, Gao X, Fischer C, Miller NE, Tewari K. 2004. Dissection of antiviral and
immune regulatory functions of tumor necrosis factor receptors in a chronic lymphocytic
choriomeningitis virus infection. J Virol 78: 3906-18
387. Razvi ES, Jiang Z, Woda BA, Welsh RM. 1995. Lymphocyte apoptosis during the
silencing of the immune response to acute viral infections in normal, lpr, and Bcl-2-
transgenic mice. Am J Pathol 147: 79-91
388. Zimmermann C, Rawiel M, Blaser C, Kaufmann M, Pircher H. 1996. Homeostatic
regulation of CD8+ T cells after antigen challenge in the absence of Fas (CD95). Eur J
Immunol 26: 2903-10
389. Weant AE, Michalek RD, Khan IU, Holbrook BC, Willingham MC, Grayson JM. 2008.
Apoptosis regulators Bim and Fas function concurrently to control autoimmunity and
CD8+ T cell contraction. Immunity 28: 218-30
390. Grayson JM, Weant AE, Holbrook BC, Hildeman D. 2006. Role of Bim in regulating
CD8+ T-cell responses during chronic viral infection. J Virol 80: 8627-38
138
391. La Gruta NL, Kedzierska K, Stambas J, Doherty PC. 2007. A question of self-
preservation: immunopathology in influenza virus infection. Immunol Cell Biol 85: 85-92
392. Bouvier NM, Palese P. 2008. The biology of influenza viruses. Vaccine 26 Suppl 4: D49-
53
393. Ennis FA, Verbonitz M, Reichelderfer P, Daniel S. 1976. Recombination of influenza A
virus strains: effect on pathogenicity. Dev Biol Stand 33: 220-5
394. Thomas PG, Keating R, Hulse-Post DJ, Doherty PC. 2006. Cell-mediated protection in
influenza infection. Emerg Infect Dis 12: 48-54
395. Iwasaki A, Pillai PS. 2014. Innate immunity to influenza virus infection. Nat Rev
Immunol 14: 315-28
396. Kohlmeier JE, Woodland DL. 2009. Immunity to respiratory viruses. Annu Rev Immunol
27: 61-82
397. Hashimoto Y, Moki T, Takizawa T, Shiratsuchi A, Nakanishi Y. 2007. Evidence for
phagocytosis of influenza virus-infected, apoptotic cells by neutrophils and macrophages
in mice. J Immunol 178: 2448-57
398. Hikono H, Kohlmeier JE, Ely KH, Scott I, Roberts AD, Blackman MA, Woodland DL.
2006. T-cell memory and recall responses to respiratory virus infections. Immunol Rev
211: 119-32
399. Legge KL, Braciale TJ. 2003. Accelerated migration of respiratory dendritic cells to the
regional lymph nodes is limited to the early phase of pulmonary infection. Immunity 18:
265-77
400. Mellman I, Steinman RM. 2001. Dendritic cells: specialized and regulated antigen
processing machines. Cell 106: 255-8
401. Moll H. 2003. Dendritic cells and host resistance to infection. Cell Microbiol 5: 493-500
402. Vermaelen KY, Carro-Muino I, Lambrecht BN, Pauwels RA. 2001. Specific migratory
dendritic cells rapidly transport antigen from the airways to the thoracic lymph nodes. J
Exp Med 193: 51-60
403. Kim TS, Braciale TJ. 2009. Respiratory dendritic cell subsets differ in their capacity to
support the induction of virus-specific cytotoxic CD8+ T cell responses. PLoS One 4:
e4204
404. Kim TS, Gorski SA, Hahn S, Murphy KM, Braciale TJ. 2014. Distinct dendritic cell
subsets dictate the fate decision between effector and memory CD8(+) T cell
differentiation by a CD24-dependent mechanism. Immunity 40: 400-13
405. McGill J, Van Rooijen N, Legge KL. 2010. IL-15 trans-presentation by pulmonary
dendritic cells promotes effector CD8 T cell survival during influenza virus infection. J
Exp Med 207: 521-34
406. Belz GT, Wodarz D, Diaz G, Nowak MA, Doherty PC. 2002. Compromised influenza
virus-specific CD8(+)-T-cell memory in CD4(+)-T-cell-deficient mice. J Virol 76:
12388-93
407. Brown DM, Dilzer AM, Meents DL, Swain SL. 2006. CD4 T cell-mediated protection
from lethal influenza: perforin and antibody-mediated mechanisms give a one-two punch.
J Immunol 177: 2888-98
408. Kim TS, Sun J, Braciale TJ. 2011. T cell responses during influenza infection: getting
and keeping control. Trends Immunol 32: 225-31
409. McKinstry KK, Strutt TM, Swain SL. 2011. Hallmarks of CD4 T cell immunity against
influenza. J Intern Med 269: 507-18
139
410. Doherty PC, Topham DJ, Tripp RA, Cardin RD, Brooks JW, Stevenson PG. 1997.
Effector CD4+ and CD8+ T-cell mechanisms in the control of respiratory virus
infections. Immunol Rev 159: 105-17
411. Oda T, Akaike T, Hamamoto T, Suzuki F, Hirano T, Maeda H. 1989. Oxygen radicals in
influenza-induced pathogenesis and treatment with pyran polymer-conjugated SOD.
Science 244: 974-6
412. Salek-Ardakani S, Croft M. 2010. Tumor necrosis factor receptor/tumor necrosis factor
family members in antiviral CD8 T-cell immunity. J Interferon Cytokine Res 30: 205-18
413. Watts TH. 2005. TNF/TNFR family members in costimulation of T cell responses. Annu
Rev Immunol 23: 23-68
414. Wortzman ME, Clouthier DL, McPherson AJ, Lin GH, Watts TH. 2013. The contextual
role of TNFR family members in CD8(+) T-cell control of viral infections. Immunol Rev
255: 125-48
415. Aggarwal BB. 2003. Signalling pathways of the TNF superfamily: a double-edged sword.
Nat Rev Immunol 3: 745-56
416. Croft M. 2009. The role of TNF superfamily members in T-cell function and diseases.
Nat Rev Immunol 9: 271-85
417. Croft M, Benedict CA, Ware CF. 2013. Clinical targeting of the TNF and TNFR
superfamilies. Nat Rev Drug Discov 12: 147-68
418. Shuford WW, Klussman K, Tritchler DD, Loo DT, Chalupny J, Siadak AW, Brown TJ,
Emswiler J, Raecho H, Larsen CP, Pearson TC, Ledbetter JA, Aruffo A, Mittler RS.
1997. 4-1BB costimulatory signals preferentially induce CD8+ T cell proliferation and
lead to the amplification in vivo of cytotoxic T cell responses. J Exp Med 186: 47-55
419. Melero I, Shuford WW, Newby SA, Aruffo A, Ledbetter JA, Hellstrom KE, Mittler RS,
Chen L. 1997. Monoclonal antibodies against the 4-1BB T-cell activation molecule
eradicate established tumors. Nat Med 3: 682-5
420. So T, Lee SW, Croft M. 2008. Immune regulation and control of regulatory T cells by
OX40 and 4-1BB. Cytokine Growth Factor Rev 19: 253-62
421. Niu L, Strahotin S, Hewes B, Zhang B, Zhang Y, Archer D, Spencer T, Dillehay D,
Kwon B, Chen L, Vella AT, Mittler RS. 2007. Cytokine-mediated disruption of
lymphocyte trafficking, hemopoiesis, and induction of lymphopenia, anemia, and
thrombocytopenia in anti-CD137-treated mice. J Immunol 178: 4194-213
422. Lee SW, Salek-Ardakani S, Mittler RS, Croft M. 2009. Hypercostimulation through 4-
1BB distorts homeostasis of immune cells. J Immunol 182: 6753-62
423. Zhu Y, Zhu G, Luo L, Flies AS, Chen L. 2007. CD137 stimulation delivers an antigen-
independent growth signal for T lymphocytes with memory phenotype. Blood 109: 4882-
9
424. Ascierto PA, Simeone E, Sznol M, Fu YX, Melero I. 2010. Clinical experiences with
anti-CD137 and anti-PD1 therapeutic antibodies. Semin Oncol 37: 508-16
425. Vinay DS, Kwon BS. 2012. Immunotherapy of cancer with 4-1BB. Mol Cancer Ther 11:
1062-70
426. Kwon BS, Weissman SM. 1989. cDNA sequences of two inducible T-cell genes. Proc
Natl Acad Sci U S A 86: 1963-7
427. Goodwin RG, Din WS, Davis-Smith T, Anderson DM, Gimpel SD, Sato TA,
Maliszewski CR, Brannan CI, Copeland NG, Jenkins NA, et al. 1993. Molecular cloning
140
of a ligand for the inducible T cell gene 4-1BB: a member of an emerging family of
cytokines with homology to tumor necrosis factor. Eur J Immunol 23: 2631-41
428. Alderson MR, Smith CA, Tough TW, Davis-Smith T, Armitage RJ, Falk B, Roux E,
Baker E, Sutherland GR, Din WS. 1994. Molecular and biological characterization of
human 4-1BB and its ligand. Eur J Immunol 24: 2219-27
429. Schwarz H, Valbracht J, Tuckwell J, von Kempis J, Lotz M. 1995. ILA, the human 4-
1BB homologue, is inducible in lymphoid and other cell lineages. Blood 85: 1043-52
430. Zhou Z, Kim S, Hurtado J, Lee ZH, Kim KK, Pollok KE, Kwon BS. 1995.
Characterization of human homologue of 4-1BB and its ligand. Immunol Lett 45: 67-73
431. Won EY, Cha K, Byun JS, Kim DU, Shin S, Ahn B, Kim YH, Rice AJ, Walz T, Kwon
BS, Cho HS. 2010. The structure of the trimer of human 4-1BB ligand is unique among
members of the tumor necrosis factor superfamily. J Biol Chem 285: 9202-10
432. Wang C, Lin GH, McPherson AJ, Watts TH. 2009. Immune regulation by 4-1BB and 4-
1BBL: complexities and challenges. Immunol Rev 229: 192-215
433. Lee HW, Park SJ, Choi BK, Kim HH, Nam KO, Kwon BS. 2002. 4-1BB promotes the
survival of CD8+ T lymphocytes by increasing expression of Bcl-xL and Bfl-1. J
Immunol 169: 4882-8
434. Tan JT, Ha J, Cho HR, Tucker-Burden C, Hendrix RC, Mittler RS, Pearson TC, Larsen
CP. 2000. Analysis of expression and function of the costimulatory molecule 4-1BB in
alloimmune responses. Transplantation 70: 175-83
435. Lee SW, Park Y, Song A, Cheroutre H, Kwon BS, Croft M. 2006. Functional dichotomy
between OX40 and 4-1BB in modulating effector CD8 T cell responses. J Immunol 177:
4464-72
436. Lin GH, Sedgmen BJ, Moraes TJ, Snell LM, Topham DJ, Watts TH. 2009. Endogenous
4-1BB ligand plays a critical role in protection from influenza-induced disease. J
Immunol 182: 934-47
437. Wang C, McPherson AJ, Jones RB, Kawamura KS, Lin GH, Lang PA, Ambagala T,
Pellegrini M, Calzascia T, Aidarus N, Elford AR, Yue FY, Kremmer E, Kovacs CM,
Benko E, Tremblay C, Routy JP, Bernard NF, Ostrowski MA, Ohashi PS, Watts TH.
2012. Loss of the signaling adaptor TRAF1 causes CD8+ T cell dysregulation during
human and murine chronic infection. J Exp Med 209: 77-91
438. Seo SK, Park HY, Choi JH, Kim WY, Kim YH, Jung HW, Kwon B, Lee HW, Kwon BS.
2003. Blocking 4-1BB/4-1BB ligand interactions prevents herpetic stromal keratitis. J
Immunol 171: 576-83
439. Pollok KE, Kim YJ, Hurtado J, Zhou Z, Kim KK, Kwon BS. 1994. 4-1BB T-cell antigen
binds to mature B cells and macrophages, and costimulates anti-mu-primed splenic B
cells. Eur J Immunol 24: 367-74
440. Kang YJ, Kim SO, Shimada S, Otsuka M, Seit-Nebi A, Kwon BS, Watts TH, Han J.
2007. Cell surface 4-1BBL mediates sequential signaling pathways 'downstream' of TLR
and is required for sustained TNF production in macrophages. Nat Immunol 8: 601-9
441. Futagawa T, Akiba H, Kodama T, Takeda K, Hosoda Y, Yagita H, Okumura K. 2002.
Expression and function of 4-1BB and 4-1BB ligand on murine dendritic cells. Int
Immunol 14: 275-86
442. Snell LM, Lin GH, McPherson AJ, Moraes TJ, Watts TH. 2011. T-cell intrinsic effects of
GITR and 4-1BB during viral infection and cancer immunotherapy. Immunol Rev 244:
197-217
141
443. Bertram EM, Lau P, Watts TH. 2002. Temporal segregation of 4-1BB versus CD28-
mediated costimulation: 4-1BB ligand influences T cell numbers late in the primary
response and regulates the size of the T cell memory response following influenza
infection. J Immunol 168: 3777-85
444. Lin GH, Snell LM, Wortzman ME, Clouthier DL, Watts TH. 2013. GITR-dependent
regulation of 4-1BB expression: implications for T cell memory and anti-4-1BB-induced
pathology. J Immunol 190: 4627-39
445. Lin GH, Edele F, Mbanwi AN, Wortzman ME, Snell LM, Vidric M, Roth K, Hauser AE,
Watts TH. 2012. Contribution of 4-1BBL on radioresistant cells in providing survival
signals through 4-1BB expressed on CD8(+) memory T cells in the bone marrow. Eur J
Immunol 42: 2861-74
446. Kwon BS, Hurtado JC, Lee ZH, Kwack KB, Seo SK, Choi BK, Koller BH, Wolisi G,
Broxmeyer HE, Vinay DS. 2002. Immune responses in 4-1BB (CD137)-deficient mice. J
Immunol 168: 5483-90
447. Tan JT, Whitmire JK, Ahmed R, Pearson TC, Larsen CP. 1999. 4-1BB ligand, a member
of the TNF family, is important for the generation of antiviral CD8 T cell responses. J
Immunol 163: 4859-68
448. Hendriks J, Xiao Y, Rossen JW, van der Sluijs KF, Sugamura K, Ishii N, Borst J. 2005.
During viral infection of the respiratory tract, CD27, 4-1BB, and OX40 collectively
determine formation of CD8+ memory T cells and their capacity for secondary
expansion. J Immunol 175: 1665-76
449. Taraban VY, Rowley TF, O'Brien L, Chan HT, Haswell LE, Green MH, Tutt AL,
Glennie MJ, Al-Shamkhani A. 2002. Expression and costimulatory effects of the TNF
receptor superfamily members CD134 (OX40) and CD137 (4-1BB), and their role in the
generation of anti-tumor immune responses. Eur J Immunol 32: 3617-27
450. Wen T, Bukczynski J, Watts TH. 2002. 4-1BB ligand-mediated costimulation of human
T cells induces CD4 and CD8 T cell expansion, cytokine production, and the
development of cytolytic effector function. J Immunol 168: 4897-906
451. Pulle G, Vidric M, Watts TH. 2006. IL-15-dependent induction of 4-1BB promotes
antigen-independent CD8 memory T cell survival. J Immunol 176: 2739-48
452. Rouse BT, Sarangi PP, Suvas S. 2006. Regulatory T cells in virus infections. Immunol
Rev 212: 272-86
453. Keynan Y, Card CM, McLaren PJ, Dawood MR, Kasper K, Fowke KR. 2008. The role of
regulatory T cells in chronic and acute viral infections. Clin Infect Dis 46: 1046-52
454. Zheng G, Wang B, Chen A. 2004. The 4-1BB costimulation augments the proliferation of
CD4+CD25+ regulatory T cells. J Immunol 173: 2428-34
455. Choi BK, Bae JS, Choi EM, Kang WJ, Sakaguchi S, Vinay DS, Kwon BS. 2004. 4-1BB-
dependent inhibition of immunosuppression by activated CD4+CD25+ T cells. J Leukoc
Biol 75: 785-91
456. Elpek KG, Yolcu ES, Franke DD, Lacelle C, Schabowsky RH, Shirwan H. 2007. Ex vivo
expansion of CD4+CD25+FoxP3+ T regulatory cells based on synergy between IL-2 and
4-1BB signaling. J Immunol 179: 7295-304
457. Irie J, Wu Y, Kachapati K, Mittler RS, Ridgway WM. 2007. Modulating protective and
pathogenic CD4+ subsets via CD137 in type 1 diabetes. Diabetes 56: 186-96
142
458. Robertson SJ, Messer RJ, Carmody AB, Mittler RS, Burlak C, Hasenkrug KJ. 2008.
CD137 costimulation of CD8+ T cells confers resistance to suppression by virus-induced
regulatory T cells. J Immunol 180: 5267-74
459. Hippen KL, Harker-Murray P, Porter SB, Merkel SC, Londer A, Taylor DK, Bina M,
Panoskaltsis-Mortari A, Rubinstein P, Van Rooijen N, Golovina TN, Suhoski MM,
Miller JS, Wagner JE, June CH, Riley JL, Blazar BR. 2008. Umbilical cord blood
regulatory T-cell expansion and functional effects of tumor necrosis factor receptor
family members OX40 and 4-1BB expressed on artificial antigen-presenting cells. Blood
112: 2847-57
460. Choi BK, Kim YH, Kwon PM, Lee SC, Kang SW, Kim MS, Lee MJ, Kwon BS. 2009. 4-
1BB functions as a survival factor in dendritic cells. J Immunol 182: 4107-15
461. Lee SW, Park Y, Eun SY, Madireddi S, Cheroutre H, Croft M. 2012. Cutting edge: 4-
1BB controls regulatory activity in dendritic cells through promoting optimal expression
of retinal dehydrogenase. J Immunol 189: 2697-701
462. Wilcox RA, Chapoval AI, Gorski KS, Otsuji M, Shin T, Flies DB, Tamada K, Mittler
RS, Tsuchiya H, Pardoll DM, Chen L. 2002. Cutting edge: Expression of functional
CD137 receptor by dendritic cells. J Immunol 168: 4262-7
463. Zhang B, Zhang Y, Niu L, Vella AT, Mittler RS. 2010. Dendritic cells and Stat3 are
essential for CD137-induced CD8 T cell activation-induced cell death. J Immunol 184:
4770-8
464. Kuang Y, Weng X, Liu X, Zhu H, Chen Z, Chen H. 2012. Effects of 4-1BB signaling on
the biological function of murine dendritic cells. Oncol Lett 3: 477-81
465. Zhang X, Voskens CJ, Sallin M, Maniar A, Montes CL, Zhang Y, Lin W, Li G, Burch E,
Tan M, Hertzano R, Chapoval AI, Tamada K, Gastman BR, Schulze DH, Strome SE.
2010. CD137 promotes proliferation and survival of human B cells. J Immunol 184: 787-
95
466. Cole SL, Benam KH, McMichael AJ, Ho LP. 2014. Involvement of the 4-1BB/4-1BBL
Pathway in Control of Monocyte Numbers by Invariant NKT Cells. J Immunol
467. Melero I, Johnston JV, Shufford WW, Mittler RS, Chen L. 1998. NK1.1 cells express 4-
1BB (CDw137) costimulatory molecule and are required for tumor immunity elicited by
anti-4-1BB monoclonal antibodies. Cell Immunol 190: 167-72
468. Wilcox RA, Tamada K, Strome SE, Chen L. 2002. Signaling through NK cell-associated
CD137 promotes both helper function for CD8+ cytolytic T cells and responsiveness to
IL-2 but not cytolytic activity. J Immunol 169: 4230-6
469. Kim DH, Chang WS, Lee YS, Lee KA, Kim YK, Kwon BS, Kang CY. 2008. 4-1BB
engagement costimulates NKT cell activation and exacerbates NKT cell ligand-induced
airway hyperresponsiveness and inflammation. J Immunol 180: 2062-8
470. Lee SW, Park Y, So T, Kwon BS, Cheroutre H, Mittler RS, Croft M. 2008. Identification
of regulatory functions for 4-1BB and 4-1BBL in myelopoiesis and the development of
dendritic cells. Nat Immunol 9: 917-26
471. Nishimoto H, Lee SW, Hong H, Potter KG, Maeda-Yamamoto M, Kinoshita T,
Kawakami Y, Mittler RS, Kwon BS, Ware CF, Croft M, Kawakami T. 2005.
Costimulation of mast cells by 4-1BB, a member of the tumor necrosis factor receptor
superfamily, with the high-affinity IgE receptor. Blood 106: 4241-8
143
472. Lee SC, Ju SA, Pack HN, Heo SK, Suh JH, Park SM, Choi BK, Kwon BS, Kim BS.
2005. 4-1BB (CD137) is required for rapid clearance of Listeria monocytogenes
infection. Infect Immun 73: 5144-51
473. Heinisch IV, Daigle I, Knopfli B, Simon HU. 2000. CD137 activation abrogates
granulocyte-macrophage colony-stimulating factor-mediated anti-apoptosis in
neutrophils. Eur J Immunol 30: 3441-6
474. Zhang B, Maris CH, Foell J, Whitmire J, Niu L, Song J, Kwon BS, Vella AT, Ahmed R,
Jacob J, Mittler RS. 2007. Immune suppression or enhancement by CD137 T cell
costimulation during acute viral infection is time dependent. J Clin Invest 117: 3029-41
475. Chung JY, Park YC, Ye H, Wu H. 2002. All TRAFs are not created equal: common and
distinct molecular mechanisms of TRAF-mediated signal transduction. J Cell Sci 115:
679-88
476. Silke J, Brink R. 2010. Regulation of TNFRSF and innate immune signalling complexes
by TRAFs and cIAPs. Cell Death Differ 17: 35-45
477. Cabal-Hierro L, Lazo PS. 2012. Signal transduction by tumor necrosis factor receptors.
Cell Signal 24: 1297-305
478. Rothe M, Pan MG, Henzel WJ, Ayres TM, Goeddel DV. 1995. The TNFR2-TRAF
signaling complex contains two novel proteins related to baculoviral inhibitor of
apoptosis proteins. Cell 83: 1243-52
479. Xie P. 2013. TRAF molecules in cell signaling and in human diseases. J Mol Signal 8: 7
480. Ely KR, Kodandapani R, Wu S. 2007. Protein-protein interactions in TRAF3. Adv Exp
Med Biol 597: 114-21
481. Chung JY, Lu M, Yin Q, Lin SC, Wu H. 2007. Molecular basis for the unique specificity
of TRAF6. Adv Exp Med Biol 597: 122-30
482. Lee HW, Nam KO, Park SJ, Kwon BS. 2003. 4-1BB enhances CD8+ T cell expansion by
regulating cell cycle progression through changes in expression of cyclins D and E and
cyclin-dependent kinase inhibitor p27kip1. Eur J Immunol 33: 2133-41
483. Lee do Y, Choi BK, Lee DG, Kim YH, Kim CH, Lee SJ, Kwon BS. 2013. 4-1BB
signaling activates the t cell factor 1 effector/beta-catenin pathway with delayed kinetics
via ERK signaling and delayed PI3K/AKT activation to promote the proliferation of
CD8+ T Cells. PLoS One 8: e69677
484. Starck L, Scholz C, Dorken B, Daniel PT. 2005. Costimulation by CD137/4-1BB inhibits
T cell apoptosis and induces Bcl-xL and c-FLIP(short) via phosphatidylinositol 3-kinase
and AKT/protein kinase B. Eur J Immunol 35: 1257-66
485. McPherson AJ, Snell LM, Mak TW, Watts TH. 2012. Opposing roles for TRAF1 in the
alternative versus classical NF-kappaB pathway in T cells. J Biol Chem 287: 23010-9
486. Sabbagh L, Pulle G, Liu Y, Tsitsikov EN, Watts TH. 2008. ERK-dependent Bim
modulation downstream of the 4-1BB-TRAF1 signaling axis is a critical mediator of CD8
T cell survival in vivo. J Immunol 180: 8093-101
487. Sabbagh L, Andreeva D, Laramee GD, Oussa NA, Lew D, Bisson N, Soumounou Y,
Pawson T, Watts TH. 2013. Leukocyte-specific protein 1 links TNF receptor-associated
factor 1 to survival signaling downstream of 4-1BB in T cells. J Leukoc Biol 93: 713-21
488. Sabbagh L, Srokowski CC, Pulle G, Snell LM, Sedgmen BJ, Liu Y, Tsitsikov EN, Watts
TH. 2006. A critical role for TNF receptor-associated factor 1 and Bim down-regulation
in CD8 memory T cell survival. Proc Natl Acad Sci U S A 103: 18703-8
144
489. Zarnegar BJ, Wang Y, Mahoney DJ, Dempsey PW, Cheung HH, He J, Shiba T, Yang X,
Yeh WC, Mak TW, Korneluk RG, Cheng G. 2008. Noncanonical NF-kappaB activation
requires coordinated assembly of a regulatory complex of the adaptors cIAP1, cIAP2,
TRAF2 and TRAF3 and the kinase NIK. Nat Immunol 9: 1371-8
490. Vallabhapurapu S, Matsuzawa A, Zhang W, Tseng PH, Keats JJ, Wang H, Vignali DA,
Bergsagel PL, Karin M. 2008. Nonredundant and complementary functions of TRAF2
and TRAF3 in a ubiquitination cascade that activates NIK-dependent alternative NF-
kappaB signaling. Nat Immunol 9: 1364-70
491. Zheng C, Kabaleeswaran V, Wang Y, Cheng G, Wu H. 2010. Crystal structures of the
TRAF2: cIAP2 and the TRAF1: TRAF2: cIAP2 complexes: affinity, specificity, and
regulation. Mol Cell 38: 101-13
492. Zapata JM, Krajewska M, Krajewski S, Kitada S, Welsh K, Monks A, McCloskey N,
Gordon J, Kipps TJ, Gascoyne RD, Shabaik A, Reed JC. 2000. TNFR-associated factor
family protein expression in normal tissues and lymphoid malignancies. J Immunol 165:
5084-96
493. Arron JR, Pewzner-Jung Y, Walsh MC, Kobayashi T, Choi Y. 2002. Regulation of the
subcellular localization of tumor necrosis factor receptor-associated factor (TRAF)2 by
TRAF1 reveals mechanisms of TRAF2 signaling. J Exp Med 196: 923-34
494. Wicovsky A, Henkler F, Salzmann S, Scheurich P, Kneitz C, Wajant H. 2009. Tumor
necrosis factor receptor-associated factor-1 enhances proinflammatory TNF receptor-2
signaling and modifies TNFR1-TNFR2 cooperation. Oncogene 28: 1769-81
495. Madireddi S, Eun SY, Lee SW, Nemcovicova I, Mehta AK, Zajonc DM, Nishi N, Niki T,
Hirashima M, Croft M. 2014. Galectin-9 controls the therapeutic activity of 4-1BB-
targeting antibodies. J Exp Med 211: 1433-48
496. Schwarz H, Tuckwell J, Lotz M. 1993. A receptor induced by lymphocyte activation
(ILA): a new member of the human nerve-growth-factor/tumor-necrosis-factor receptor
family. Gene 134: 295-8
497. Jiang D, Chen Y, Schwarz H. 2008. CD137 induces proliferation of murine
hematopoietic progenitor cells and differentiation to macrophages. J Immunol 181: 3923-
32
498. Jiang D, Yue PS, Drenkard D, Schwarz H. 2008. Induction of proliferation and
monocytic differentiation of human CD34+ cells by CD137 ligand signaling. Stem Cells
26: 2372-81
499. Cannons JL, Lau P, Ghumman B, DeBenedette MA, Yagita H, Okumura K, Watts TH.
2001. 4-1BB ligand induces cell division, sustains survival, and enhances effector
function of CD4 and CD8 T cells with similar efficacy. J Immunol 167: 1313-24
500. Takahashi C, Mittler RS, Vella AT. 1999. Cutting edge: 4-1BB is a bona fide CD8 T cell
survival signal. J Immunol 162: 5037-40
501. Lin GH, Liu Y, Ambagala T, Kwon BS, Ohashi PS, Watts TH. 2010. Evaluating the
cellular targets of anti-4-1BB agonist antibody during immunotherapy of a pre-
established tumor in mice. PLoS One 5: e11003
502. Moraes TJ, Lin GH, Wen T, Watts TH. 2011. Incorporation of 4-1BB ligand into an
adenovirus vaccine vector increases the number of functional antigen-specific CD8 T
cells and enhances the duration of protection against influenza-induced respiratory
disease. Vaccine 29: 6301-12
145
503. Steinman RM. 2007. Dendritic cells: understanding immunogenicity. Eur J Immunol 37
Suppl 1: S53-60
504. Summers deLuca L, Gommerman JL. 2012. Fine-tuning of dendritic cell biology by the
TNF superfamily. Nat Rev Immunol 12: 339-51
505. Lutz MB, Kukutsch N, Ogilvie AL, Rossner S, Koch F, Romani N, Schuler G. 1999. An
advanced culture method for generating large quantities of highly pure dendritic cells
from mouse bone marrow. J Immunol Methods 223: 77-92
506. Bertram EM, Dawicki W, Sedgmen B, Bramson JL, Lynch DH, Watts TH. 2004. A
switch in costimulation from CD28 to 4-1BB during primary versus secondary CD8 T
cell response to influenza in vivo. J Immunol 172: 981-8
507. Halstead ES, Mueller YM, Altman JD, Katsikis PD. 2002. In vivo stimulation of CD137
broadens primary antiviral CD8+ T cell responses. Nat Immunol 3: 536-41
508. Salih HR, Schmetzer HM, Burke C, Starling GC, Dunn R, Pelka-Fleischer R, Nuessler V,
Kiener PA. 2001. Soluble CD137 (4-1BB) ligand is released following leukocyte
activation and is found in sera of patients with hematological malignancies. J Immunol
167: 4059-66
509. Liu GZ, Gomes AC, Putheti P, Karrenbauer V, Kostulas K, Press R, Hillert J,
Hjelmstrom P, Gao XG. 2006. Increased soluble 4-1BB ligand (4-1BBL) levels in
peripheral blood of patients with multiple sclerosis. Scand J Immunol 64: 412-9
510. Kuka M, Munitic I, Giardino Torchia ML, Ashwell JD. 2013. CD70 is downregulated by
interaction with CD27. J Immunol 191: 2282-9
511. Stephan MT, Ponomarev V, Brentjens RJ, Chang AH, Dobrenkov KV, Heller G,
Sadelain M. 2007. T cell-encoded CD80 and 4-1BBL induce auto- and
transcostimulation, resulting in potent tumor rejection. Nat Med 13: 1440-9
512. Day CL, Kaufmann DE, Kiepiela P, Brown JA, Moodley ES, Reddy S, Mackey EW,
Miller JD, Leslie AJ, DePierres C, Mncube Z, Duraiswamy J, Zhu B, Eichbaum Q,
Altfeld M, Wherry EJ, Coovadia HM, Goulder PJ, Klenerman P, Ahmed R, Freeman GJ,
Walker BD. 2006. PD-1 expression on HIV-specific T cells is associated with T-cell
exhaustion and disease progression. Nature 443: 350-4
513. Boni C, Fisicaro P, Valdatta C, Amadei B, Di Vincenzo P, Giuberti T, Laccabue D,
Zerbini A, Cavalli A, Missale G, Bertoletti A, Ferrari C. 2007. Characterization of
hepatitis B virus (HBV)-specific T-cell dysfunction in chronic HBV infection. J Virol 81:
4215-25
514. Radziewicz H, Ibegbu CC, Fernandez ML, Workowski KA, Obideen K, Wehbi M,
Hanson HL, Steinberg JP, Masopust D, Wherry EJ, Altman JD, Rouse BT, Freeman GJ,
Ahmed R, Grakoui A. 2007. Liver-infiltrating lymphocytes in chronic human hepatitis C
virus infection display an exhausted phenotype with high levels of PD-1 and low levels of
CD127 expression. J Virol 81: 2545-53
515. Freeman GJ, Wherry EJ, Ahmed R, Sharpe AH. 2006. Reinvigorating exhausted HIV-
specific T cells via PD-1-PD-1 ligand blockade. J Exp Med 203: 2223-7
516. Wilson EB, Brooks DG. 2011. The role of IL-10 in regulating immunity to persistent
viral infections. Curr Top Microbiol Immunol 350: 39-65
517. Kim PS, Ahmed R. 2010. Features of responding T cells in cancer and chronic infection.
Curr Opin Immunol 22: 223-30
146
518. Zinkernagel RM, Planz O, Ehl S, Battegay M, Odermatt B, Klenerman P, Hengartner H.
1999. General and specific immunosuppression caused by antiviral T-cell responses.
Immunol Rev 168: 305-15
519. Moskophidis D, Lechner F, Pircher H, Zinkernagel RM. 1993. Virus persistence in
acutely infected immunocompetent mice by exhaustion of antiviral cytotoxic effector T
cells. Nature 362: 758-61
520. Cerwenka A, Lanier LL. 2001. Ligands for natural killer cell receptors: redundancy or
specificity. Immunol Rev 181: 158-69
521. Battegay M, Cooper S, Althage A, Banziger J, Hengartner H, Zinkernagel RM. 1991.
Quantification of lymphocytic choriomeningitis virus with an immunological focus assay
in 24- or 96-well plates. J Virol Methods 33: 191-8
522. Haworth O, Cernadas M, Levy BD. 2011. NK cells are effectors for resolvin E1 in the
timely resolution of allergic airway inflammation. J Immunol 186: 6129-35
523. Cupedo T, Mebius RE. 2005. Cellular interactions in lymph node development. J
Immunol 174: 21-5
524. Finke D. 2005. Fate and function of lymphoid tissue inducer cells. Curr Opin Immunol
17: 144-50
525. Jewett A, Bonavida B. 1995. Interferon-alpha activates cytotoxic function but inhibits
interleukin-2-mediated proliferation and tumor necrosis factor-alpha secretion by
immature human natural killer cells. J Clin Immunol 15: 35-44
526. Loza MJ, Perussia B. 2004. Differential regulation of NK cell proliferation by type I and
type II IFN. Int Immunol 16: 23-32
527. Nguyen KB, Salazar-Mather TP, Dalod MY, Van Deusen JB, Wei XQ, Liew FY,
Caligiuri MA, Durbin JE, Biron CA. 2002. Coordinated and distinct roles for IFN-alpha
beta, IL-12, and IL-15 regulation of NK cell responses to viral infection. J Immunol 169:
4279-87
528. Navarini AA, Recher M, Lang KS, Georgiev P, Meury S, Bergthaler A, Flatz L, Bille J,
Landmann R, Odermatt B, Hengartner H, Zinkernagel RM. 2006. Increased susceptibility
to bacterial superinfection as a consequence of innate antiviral responses. Proc Natl Acad
Sci U S A 103: 15535-9
529. Miko I, Brath E, Nemeth N, Furka A, Sipka S, Jr., Peto K, Serfozo J, Kovacs J, Imre S,
Benko I, Galuska L, Sipka S, Acs G, Furka I. 2007. Spleen autotransplantation.
Morphological and functional follow-up after spleen autotransplantation in mice: a
research summary. Microsurgery 27: 312-6
530. Yamataka A, Fujiwara T, Tsuchioka T, Kurosu Y, Sunagawa M. 1996. Heterotopic
splenic autotransplantation in a neonate with splenic rupture, leading to normal splenic
function. J Pediatr Surg 31: 239-40
531. Tavassoli M. 1975. Limitation of splenic growth as studied by heterotopic splenic
implants. Blood 46: 631-5
532. Riera M, Buczacki S, Khan ZA. 2009. Splenic regeneration following splenectomy and
impact on sepsis: a clinical review. J R Soc Med 102: 139-42
533. Tan JK, Watanabe T. 2014. Murine spleen tissue regeneration from neonatal spleen
capsule requires lymphotoxin priming of stromal cells. J Immunol 193: 1194-203
534. Metcalf D. 1964. RESTRICTED GROWTH CAPACITY OF MULTIPLE SPLEEN
GRAFTS. Transplantation 2: 387-92
147
535. Metcalf D. 1963. Spleen graft growth in splenectomised mice. Aust J Exp Biol Med Sci
41: 51-60
536. Janssen EM, Lemmens EE, Wolfe T, Christen U, von Herrath MG, Schoenberger SP.
2003. CD4+ T cells are required for secondary expansion and memory in CD8+ T
lymphocytes. Nature 421: 852-6
537. Feau S, Arens R, Togher S, Schoenberger SP. 2011. Autocrine IL-2 is required for
secondary population expansion of CD8(+) memory T cells. Nat Immunol 12: 908-13
538. Janssen EM, Droin NM, Lemmens EE, Pinkoski MJ, Bensinger SJ, Ehst BD, Griffith TS,
Green DR, Schoenberger SP. 2005. CD4+ T-cell help controls CD8+ T-cell memory via
TRAIL-mediated activation-induced cell death. Nature 434: 88-93
539. Munitic I, Kuka M, Allam A, Scoville JP, Ashwell JD. 2013. CD70 deficiency impairs
effector CD8 T cell generation and viral clearance but is dispensable for the recall
response to lymphocytic choriomeningitis virus. J Immunol 190: 1169-79
540. Kruglov AA, Lampropoulou V, Fillatreau S, Nedospasov SA. 2011. Pathogenic and
protective functions of TNF in neuroinflammation are defined by its expression in T
lymphocytes and myeloid cells. J Immunol 187: 5660-70
541. Winsauer C, Kruglov AA, Chashchina AA, Drutskaya MS, Nedospasov SA. 2013.
Cellular sources of pathogenic and protective TNF and experimental strategies based on
utilization of TNF humanized mice. Cytokine Growth Factor Rev
542. Clouthier DL, Watts TH. 2014. Cell-specific and context-dependent effects of GITR in
cancer, autoimmunity, and infection. Cytokine Growth Factor Rev
543. Juhasz K, Buzas K, Duda E. 2013. Importance of reverse signaling of the TNF
superfamily in immune regulation. Expert Rev Clin Immunol 9: 335-48
544. Shao Z, Schwarz H. 2011. CD137 ligand, a member of the tumor necrosis factor family,
regulates immune responses via reverse signal transduction. J Leukoc Biol 89: 21-9
545. Sun M, Fink PJ. 2007. A new class of reverse signaling costimulators belongs to the TNF
family. J Immunol 179: 4307-12
546. Eissner G, Kolch W, Scheurich P. 2004. Ligands working as receptors: reverse signaling
by members of the TNF superfamily enhance the plasticity of the immune system.
Cytokine Growth Factor Rev 15: 353-66
547. Sollner L, Shaqireen DOKMM, Wu JT, Schwarz H. 2007. Signal transduction
mechanisms of CD137 ligand in human monocytes. Cell Signal 19: 1899-908
548. Saito K, Ohara N, Hotokezaka H, Fukumoto S, Yuasa K, Naito M, Fujiwara T,
Nakayama K. 2004. Infection-induced up-regulation of the costimulatory molecule 4-
1BB in osteoblastic cells and its inhibitory effect on M-CSF/RANKL-induced in vitro
osteoclastogenesis. J Biol Chem 279: 13555-63
549. Watts AD, Hunt NH, Wanigasekara Y, Bloomfield G, Wallach D, Roufogalis BD,
Chaudhri G. 1999. A casein kinase I motif present in the cytoplasmic domain of members
of the tumour necrosis factor ligand family is implicated in 'reverse signalling'. EMBO J
18: 2119-26
550. Sun M, Lee S, Karray S, Levi-Strauss M, Ames KT, Fink PJ. 2007. Cutting edge: two
distinct motifs within the Fas ligand tail regulate Fas ligand-mediated costimulation. J
Immunol 179: 5639-43
551. Ma J, Bang BR, Lu J, Eun SY, Otsuka M, Croft M, Tobias P, Han J, Takeuchi O, Akira
S, Karin M, Yagita H, Kang YJ. 2013. The TNF family member 4-1BBL sustains
148
inflammation by interacting with TLR signaling components during late-phase activation.
Sci Signal 6: ra87
552. Moh MC, Lorenzini PA, Gullo C, Schwarz H. 2013. Tumor necrosis factor receptor 1
associates with CD137 ligand and mediates its reverse signaling. FASEB J 27: 2957-66
553. Bae JS, Choi JK, Moon JH, Kim EC, Croft M, Lee HW. 2012. Novel transmembrane
protein 126A (TMEM126A) couples with CD137L reverse signals in myeloid cells. Cell
Signal 24: 2227-36
554. Narni-Mancinelli E, Jaeger BN, Bernat C, Fenis A, Kung S, De Gassart A, Mahmood S,
Gut M, Heath SC, Estelle J, Bertosio E, Vely F, Gastinel LN, Beutler B, Malissen B,
Malissen M, Gut IG, Vivier E, Ugolini S. 2012. Tuning of natural killer cell reactivity by
NKp46 and Helios calibrates T cell responses. Science 335: 344-8
555. Koller BH, Marrack P, Kappler JW, Smithies O. 1990. Normal development of mice
deficient in beta 2M, MHC class I proteins, and CD8+ T cells. Science 248: 1227-30
556. Gonzalez-Cabrero J, Wise CJ, Latchman Y, Freeman GJ, Sharpe AH, Reiser H. 1999.
CD48-deficient mice have a pronounced defect in CD4(+) T cell activation. Proc Natl
Acad Sci U S A 96: 1019-23
557. Cerwenka A, Bakker AB, McClanahan T, Wagner J, Wu J, Phillips JH, Lanier LL. 2000.
Retinoic acid early inducible genes define a ligand family for the activating NKG2D
receptor in mice. Immunity 12: 721-7
558. Lodoen M, Ogasawara K, Hamerman JA, Arase H, Houchins JP, Mocarski ES, Lanier
LL. 2003. NKG2D-mediated natural killer cell protection against cytomegalovirus is
impaired by viral gp40 modulation of retinoic acid early inducible 1 gene molecules. J
Exp Med 197: 1245-53
559. Ogasawara K, Hamerman JA, Hsin H, Chikuma S, Bour-Jordan H, Chen T, Pertel T,
Carnaud C, Bluestone JA, Lanier LL. 2003. Impairment of NK cell function by NKG2D
modulation in NOD mice. Immunity 18: 41-51
560. Kamizono S, Duncan GS, Seidel MG, Morimoto A, Hamada K, Grosveld G, Akashi K,
Lind EF, Haight JP, Ohashi PS, Look AT, Mak TW. 2009. Nfil3/E4bp4 is required for
the development and maturation of NK cells in vivo. J Exp Med 206: 2977-86
561. Diana J, Lehuen A. 2009. NKT cells: friend or foe during viral infections? Eur J Immunol
39: 3283-91
562. Diana J, Griseri T, Lagaye S, Beaudoin L, Autrusseau E, Gautron AS, Tomkiewicz C,
Herbelin A, Barouki R, von Herrath M, Dalod M, Lehuen A. 2009. NKT cell-
plasmacytoid dendritic cell cooperation via OX40 controls viral infection in a tissue-
specific manner. Immunity 30: 289-99
563. Pearson HA, Johnston D, Smith KA, Touloukian RJ. 1978. The born-again spleen.
Return of splenic function after splenectomy for trauma. N Engl J Med 298: 1389-92
564. Ludtke FE, Mack SC, Schuff-Werner P, Voth E. 1989. Splenic function after
splenectomy for trauma. Role of autotransplantation and splenosis. Acta Chir Scand 155:
533-9
565. Dodds WJ, Taylor AJ, Erickson SJ, Stewart ET, Lawson TL. 1990. Radiologic imaging
of splenic anomalies. AJR Am J Roentgenol 155: 805-10
566. Curtis GM, Movitz D. 1946. The surgical significance of the accessory spleen. Ann Surg
123: 276-98
567. Khan ZA, Dikki PE. 2004. Return of a normal functioning spleen after traumatic
splenectomy. J R Soc Med 97: 391-2
149
568. Zoli G, Corazza GR, D'Amato G, Bartoli R, Baldoni F, Gasbarrini G. 1994. Splenic
autotransplantation after splenectomy: tuftsin activity correlates with residual splenic
function. Br J Surg 81: 716-8
569. Marine D, Manley OT. 1920. HOMEOTRANSPLANTATION AND
AUTOTRANSPLANTATION OF THE SPLEEN IN RABBITS : III. FURTHER DATA
ON GROWTH, PERMANENCE, EFFECT OF AGE, AND PARTIAL OR COMPLETE
REMOVAL OF THE SPLEEN. J Exp Med 32: 113-33