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Cancer Immunotherapy Targeting
Cancer and Innate Immune Cells
Hun Sik Kim
University of Ulsan
College of Medicine
2011 춘계면역학회
Problems of traditional cancer therapies
Many therapies are aiming at stopping or slowing the growth of tumor, but not eradicating it. This may lead to long-term remission with developing a resistance to the drug.
Moreover, chemotherapy is nearly of no use to control metastasis and involves a series of side effects including disruption of the body’s immune system.
Contribution of the immune system to the
success of anti-tumor therapy
The importance of immune system for the efficacy of anti-tumor radiotherapy in mice is shown.
More frequent tumor (both spontaneous and carcinogen-induced) development in immunodeficient mice.
Harnessing the immune system to treat cancer
Developing approaches to manipulate conventional cancer
therapies to prime immunity is now an exciting area of
investigation.
Boosting the immune system to battle cancer
In order to completely eradicate cancer cells, the immune system must be activated at a stage at which the tumor has been reduced to a minimum using conventional therapy.
Consideration of immunomodulatory effects of
dying cells associated with traditional therapies
Chemotherapy and radiotherapy can target tumor cells
for apoptotic destruction.
Apoptotic cells lead to immunosuppression after uptake
by macrophages, while necrotic cells induce
inflammatory responses.
Therefore, chemotherapeutic agents able to induce
immunogenic cancer cell death have a great potential to
optimise anticancer immunotherapy.
Modulation of the immune system by dying cells
What receptor is responsible for sensing DAMPs to induce pro-inflammatory response?
DAMPs: damage-associated
molecular patterns
Nat Rev Immunol, 2008
Toll-like receptor 2
senses -cell death
and contributes to
the initiation of
autoimmune diabetes
Kim et al., Immunity 27,
321-333 August 2007
Cover Paper
Essential role of TLR2 but not TLR4 in pro-
inflammatory response after primary or secondary
necrotic cell encounter
0
1000
2000
3000
4000TLR2+/+
TLR2-/-
UV STZ Eto FT PN PGN LPS
SN
TN
F-
(p
g/m
l)
0
200
400
600
800
1000TLR2+/+
TLR2-/-
_SN PNApop
Primary MEF
TN
F-
(p
g/m
l)
Does antigen presenting cells from TLR KO mouse
affect antigen-specific T cell proliferation in vivo?
SN cells trigger TLR2-dependent stimulation of
APC and priming of antigen-specific T cells in PLN
TLR2
HMGB1 mediates endogenous TLR2 activation and brain tumor regression,
Curtin et al., PLOS Med, 2009, 6, e1000010.
Carcinoma-produced factors activate myeloid cells through TLR2 to stimulate
metastasis, Kim et al., Nature, 2009, 457, 102.
Membrane-associated Hsp72 from tumor-derived exosomes mediates STAT3-
dependent immunosuppressive function of mouse and human myeloid-
derived suppressor cells, Chalmin et al., J Clin Invest, 2010, 120, 457.
High-mobility group box 1 is involved in the initial events of early loss of
transplanted islets in mice, Matsuoka et al., J Clin Invest, 2010, JCI41360.
TLR4
Toll-like receptor 4-dependent contribution of the immune system to
anticancer chemotherapy and radiotherapy, Apetoh et al., Nature Med, 2007,
13, 1050.
CD36 ligands promote sterile inflammation through assembly of a Toll-like
receptor 4 and 6 heterodimer, Stewart et al., Nature Immunol, 2010, 11, 155.
Immunogenic cell death associated with
stimulation of Toll-like receptors
Induction of immunogenic cell death and its use in
cancer immunotherapy-focusing on TLR signaling
Cancer therapy Autoimmune diseases
Alzheimer’s disease
Allograft rejection
Ischemia injury
Development of innate
and adaptive immunity
Boosting
immunity
by targeting
TLR and its
associated
signaling
Gasser, S. et al. 2006 Cancer Res., 66, 3959
Tumor cells responding to chemotherapy or radiotherapy
express ‘danger’ signals to stimulate innate killer cells
NK cell functions
Natural killer (NK) cells are lymphocytes of the innate immune system that are able to kill tumor cells and infected cells without prior sensitization.
NK cells regulate adaptive immune responses by activating DCs, macrophages and T cells.
Eric Vivier et al. 2008 Nature Immunol., 9, 503
NK cell effector functions
NK cells as a promising therapeutic
target in human cancer
Cancer stem cell – Drug resistant and radioresistant
NK cells recognize and kill human glioblastoma cells with stem cell-like
properties, Castriconi et al., J Immunol, 2009, 182, 3530.
Primitive quiescent CD34+ cells in chronic myeloid leukemia are targeted
by in vitro expanded NK cells, which are functionally enhanced by
bortezomib, Yong et al., Blood, 2009, 113, 875.
NK cells kill human melanoma cells with characteristics of cancer stem
cells, Pietra et al., Int Immunol, 2009, 21, 793.
Regulatory T cells – Immunosuppression
NK cells lyse T regulatory cells that expand in response to an
intracellular pathogen, Roy et al., J Immunol., 2008, 180, 1729.
Myeloid derived suppressor cells – Immunosuppression
Mononuclear myeloid-derived "suppressor" cells express RAE-1 and
activate NK cells, Nausch et al., Blood, 2008, 112, 4080.
Antagonistic effect of NK cells on alternatively activated monocytes: a
contribution of NK cells to CTL generation, Geldhof et al., Blood, 2002,
100, 4049.
Missing-self hypothesis
- NK
No Kill
Normal
NK
Kill
Cancer
MHC Class I
Karre et al. Nature 1986
Integration of inhibitory and activating NK cell
receptor signals regulates NK decision to kill
The dynamic regulation of NK cell effector function
E Vivier et al. Science 2011;331:44-49
The NK cell-target cell ‘zipper’
Eric Vivier et al. 2008 Nature Immunol., 9, 503
How signals from discrete receptors are integrated to determine functional outcome?
Inhibitory signaling via SHP-1 recruitment to ITIMs
I T I
M
KIR
Src kinase P
P
SHP
-1
P
P
SHP
-1
Activation
Slide by Carsten Watzl
ITIM consensus:
V/IxYxxL
Burshtyn et al., 1996 Immunity
MHC I
Activation receptors
Adapters Receptors
ITAMs: FcR , TCR CD16 (FcRIII)
FcR , TCR , DAP12 NCRs (NKp46, NKp44, NKp30)
DAP12 Activating KIR, Ly49, CD94/NKG2C
DAP10: DAP10 NKG2D
No adapter: 2B4
DNAM-1 (CD226)
CD2
NKp80
CD27 (TNFR family)
etc.
Integrins: L2 (LFA-1)
41 (VLA-4)
ITAM and NKG2D signaling pathway
ITAM-coupled receptor
Degranulation
Syk
ZA
P70
Y
Y
- P
P
+ LA
T
3BP2
SLP76 Vav PI3K
PLC2
PIP3 PIP2 DAG
IP3
Ca2+ Rac, Cdc42
ERK
Actin reorganization
PKC
Ras
SOS
Grb2
PI3K
PIP3
Vav1 Grb2
ERK Adhesion,
Polarization
NKG2D/
DAP10
PIP2
CrkL
Rac, Cdc42 Rap1
The multiple receptor-ligand interactions between NK cells and
target cells make it difficult to dissect the function of individual
NK receptors.
By employing insect cells as surrogate target cells, the specific
ligands of NK cell receptors can be expressed to test NK cell
activation.
Drosophila cell line as a customized target
for human NK cells
CD48
Apart from CD16 (FcRIIIA), which is sufficient for activation of
resting NK cells, natural cytotoxicity by resting NK cells is only
induced by mutual costimulation of activating receptors, such as
NKG2D and 2B4.
Bryceson YT et al.
2006 Immunol. Rev.,
214, 73
Synergy-dependent activation of natural cytotoxicity in
human resting NK cells
NKG2D and 2B4
synergy as a
platform model for
NK cell activation
- NKG2D plays a
critical role in
immunosurveillance
of certain tumor and
infected cells.
NKp46
2B4
CD2
NKG2D
DNAM-1
CD16
ADCC Natural Cytotoxicity
Degranulation
Cytokine release
Degranulation
Cytokine release
Synergy
Degranulation
100
101
102
103
104
100
101
102
103
104
0.18
100
101
102
103
104
100
101
102
103
104
1.30
100
101
102
103
104
100
101
102
103
104
13.4
100
101
102
103
104
100
101
102
103
104
0.20
100
101
102
103
104
100
101
102
103
104
0.41
100
101
102
103
104
100
101
102
103
104
19.2CD
56
CD107a
rNK alone S2-ULBP1 S2-ULBP1+CD48
S2-CD48 S2+IgGS2
100 101 102 103 104
0
20
40
60
80
100
%ofM
ax
100 101 102 103 104
0
20
40
60
80
100
%ofM
ax
100
101
102
103
104
0
20
40
60
80
100
%o
fM
ax
%fM
100
101
102
103
104
0
20
40
60
80
100
oa
x
S2-ULBP1+CD48S2-CD48
S2 S2-ULBP1
cIgGULBP1CD48
S2 Target cells
Synergistic activation of resting NK cells induced
by NKG2D and 2B4 ligands
Imaging live NK cells on lipid bilayers by
total internal reflection fluorescence (TIRF) microscopy
Imaging live NK cells on lipid bilayers by
total internal reflection fluorescence (TIRF) microscopy
ICAM-1+ULBP1+CD48
pY759-PLC 2
pY1217-PLC 2
PLC 2
0 22 5 15 0 22 5 15 0 22 5 15 min
NKG2D 2B4 NKG2D+2B4
pS473-AKT
AKT
p-ERK1
ERK1ERK2
p-ERK2
Synergistic activation of PLC-2 and ERK but not
PI3K/Akt in human resting NK cells
Critical role of Vav1 but not Vav2 or Vav3 in
NKG2D and 2B4 synergy
Vav1 is required for synergistic calcium mobilization, cytotoxicity,
and cytokine production
0 100 200 3000
50
100
150
200siControl
siVav1
siVav2
siVav3
[Ca ]
i2
+
Time (sec)
NKG2D+2B4
0
20
40
60
80
1.25:1 5:1 10:12.5:1
E:T ratio
Specific
lysis
(%)
siControl
siVav1
siVav2
siVav3
NKG2D+2B4
0
100
200
300
400siControlsiVav1
NKG2D 2B4 NKG2D
+2B4
cIgG1
IFN
-pro
duction
(pg
/ml)
0
500
1000
1500
2000siControlsiVav1
cIgG1 NKG2D 2B4 NKG2D
+2B4
MIP
-1
pro
duction
(pg/m
l)
Vav1
siCont
rol
siVav1
siVav2
siVav
3
Vav2
Vav3
Actin
c-Cbl but not Cbl-b limits NK cell activation and imposes
a requirement for synergy
Knockdown of c-Cbl but not Cbl-b potentiates calcium
mobilization, degranulation, and IFN- production by NKG2D
and 2B4 synergy
s iC
o n tro l
s ic -
Cb l
s iC
b l -b
c -C b l
C b l -b
A c tin
NKG2D 2B4 NKG2D+2B4
[Ca
]
i2+
Time (sec)
0 100 200 3000
50
100
150
200siControlsic-CblsiCbl-b
0 100 200 3000
50
100
150
200
[Ca
]
i2+
Time (sec)
0 100 200 3000
50
100
150
200
[Ca
]
i2+
Time (sec)
0
800
1600
2400
3200
S2 S2ULBP1
S2CD48
S2ULBP1CD48
siControlsic-Cbl
G
ran
zym
e B
(p
g/m
l)
0
200
400
600
800siControlsic-Cbl
cIgG1 NKG2D 2B4 NKG2D+2B4
IFN
- p
rod
uctio
n (
pg
/ml)
Increased Vav1 expression overcomes inhibition by c-Cbl
NKG2D or 2B4 alone becomes sufficient for NK cell activation by
overexpression of Vav1 or Vav1 CAAX
YFP control YFP-Vav1YFP-Vav1 CAAX
0
10
20
30
40
50
60
cIgG1 NKG2D 2B4 NKG2D+2B4
YFP controlYFP-Vav1YFP-Vav1 CAAX
Sp
ecific
lysis
(%
)
0
400
800
1200
1600
2000
S2 S2ULBP1
S2CD48
S2ULBP1CD48
YFP controlYFP-Vav1YFP-Vav1 CAAX
G
ran
zym
e B
(p
g/m
l)
Cytotoxicity Degranulation
Cellular localization
NK cell activation by synergy is regulated at the level
of Vav1 by a hierarchy of inhibitory mechanism
Synergistic activation
Activating Receptor
Activating Ligand
Vav1 Rac1
P P P
RAFT
KIR
MHC I
SHP
-1
Target Cell
Actin c-Cbl
Kim et al.,
Immunity (2010)
Dissecting the contribution of individual receptors to NK cell activation using Drosophila S2 cells expressing ligands of NK cell receptors
Screening potential drug candidates that enhance NK cell function by targeting specific NK cell receptor or key signaling molecules
Activation signal Inhibitory signal
Insect cell
CD16 CD2
LFA3
2B4
CD48 ICAM1
LFA1 KIR CD94/NKG2A
HLA-C HLA-E
Anti S2 cell
Rabbit serum
ITI M
ULBP
NKG2D
DAP10
NK cell
ITI M
ITA M
Analysis of NK cell activation with insect target cell system
Developing systems approach for complete
eradication of tumor cells
Chemotherapy
Radiotherapy
Immunotherapy
Vaccines
Tumor cell
Induction of
stress (DNA
damage)
response NKG2D-L
Stressed Cell
Killer cell
response
NK cell
CTL
response
TH1 cell
response
Bystander
activation
Boosting
immune
responses
Cancer targeting Immune cell targeting
Acknowledgements
SMC, SKKU University
Myung-Shik Lee
Eunsil Kim
Myoung Sook Han
Namjoo Cho
Sunshin Kim
Hyun Ah Lee
Moon-Kyu Lee
Kwang-Won Kim
Osaka University
Shizuo Akira
Kiyoshi Takeda
NIAID/NIH
Eric Long
Sumi Rajagopalan
Asmita Das
Catharina Gross
Dongfang Liu
Mike March
Mary Peterson
Susina Vielkind
Harvard University
Diane Mathis
Christopher Benoist
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