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Turning human natural killer cells on and off
Graham Cook
Section of Infection and Immunity
University of Leeds School of Medicine
Outline
1. Why do we need NK cells?
How they recognise target cells (including missing self)
2. What are the consequences of NK cell activation?
Effector functions and modulation of the immune response (NK-DC interactions)
3. What safeguards against damage to healthy tissue?
NK cell education/licensing; not such blunt instruments after all
4. NK cells detect and kill tumour cells; why then do we get cancer?
Mechanisms of NK cell evasion by tumours
5. Can we exploit NK cells for treatment of human disease?
NK cells as agents of cancer immunotherapy (and learning about NK cells from clinical studies)
Why do we need NK cells?
Their mode of recognition complements that of T cells
A reminder of how a cytotoxic T cell sees an infected cell…..
V
MHC class I: peptide
T-cell receptor (TCR)
T-cell
Infected
cell
CD8
Kill
No Yes Yes
• T cells cannot see MHC class I negative cells (blind to the virus) → NK cells fill this niche
• Evolutionary battles (versus pathogens) are a major driver of NK cell evolution and function
• Recognition by “missing self” (look for changes in expression of host cell molecules)
MHC class I Killer Inhibitory Receptor (KIR)
ITIM containing, recruit phosphatases Activation Ligand (e.g. MICA) Activation Receptors (e.g. NKG2D, NCRs)
Many are ITAM containing, recruit kinases
IFN
But, many viruses have evolved strategies to reduce MHC class I from the cell surface……….
V
TNF
Killer Inhibitory Receptor Activating Receptor
e.g. NKG2D
NK cell activation causes exocytosis
of cytotoxic granules containing:
1. Perforin (pore forming)
2. Granzymes (pro-apoptotic serine proteases)
Humans: GZMA, B, H, K, M
These cleave many substrates, including caspases
Induce very rapid apoptosis in target cells
Movie;
Lopez JA et al (2013) Perforin forms transient pores on the target cell
plasma membrane to facilitate rapid access of granzymes during killer cell attack.
Blood 121:2659-68.
From: Vivier E et al (2008) The functions of natural killer cells
Nature Immunology 9, 503 – 510
Activating
signals
Inhibitory
signals
Cytotoxicity
+
Chemokines
+
Cytokines
No Yes
The repertoire of NK cell activation and inhibitory receptors and their ligands
Yes
No Antibody dependent cellular cytotoxicity (ADCC) mediated by CD16
HeLa cells; obeying the missing self hypothesis
100 101 102 103 104HLA W6/32
100 101 102 103 104HLA W6/32
100 101 102 103 104HLA W6/32
β2M-siRNA
Control-siRNA
No siRNA
MHC Class I
+IFN-γ
0
10
20
30
40
50
Untrd CtrlsiRNA
β2m siRNA
IFNγ
HeLa K
illin
g (
%)
Un Con β2M IFN-γ
siRNA
Add IFN-γ then MHC and NK killing = ✓
If β2M then MHC and NK killing = ✓
MHC class I
“sliding scale”
0
20
40
60
80
100
Untrd CtrlsiRNA
β2m siRNA
IFNγ Ctrl siRNA +
IFNγ
β2m siRNA +
IFNy
TC
32
Kill
ing (
%)
Un Con β2M
+IFN-γ
+siRNA
Un Con β2M
+siRNA
Add IFN-γ, then MHC and NK killing = ✓
(siRNA shows process is β2M, i.e. MHC-dependent)
β2M, MHC, NK killing unchanged= ✗
0
20
40
60
80
100
Untrd IFNγ
SK
ES
-1 K
illin
g (
%)
Un IFN-γ
Add IFN-γ, then MHC and NK killing unchanged = ✗
Ewing’s sarcoma : disobeying the missing self hypothesis?
TC32
SKES-1
MHC class I
Threshold model
TC32 SKES
No….they don’t disobey missing self- they show existence of threshold
Mapping the activation/inhibition threshold •Generate populations of cells with differing MHC class I expression (using β2M siRNA and IFN-γ)
•Use in killing assays and express inhibition of killing relative to control siRNA treated control
•Threshold is very similar in TC32 and SK-N-MC (very similar activating ligand expression)
Holmes TD et al (2011) A human NK cell activation/inhibition threshold allows small changes in the target cell surface phenotype
to dramatically alter susceptibility to NK cells. J Immunol. 186, 1538-1535.
Small changes in target MHC
determine susceptibility
This is good and bad….......
Threshold is based on the
behaviour of an NK cell
population
What about the behaviour of
individual NK cells?
NK cells detect and kill tumour cells and virus infected cells
They produce cytokines such as IFN-gamma, GM-CSF and TNF and chemokines such as MIP1a
What else do they do?- gene expression profiling on degranulating and non-degranulating populations
Compare by microarray
CD107
CD
56
Non-responders NR (not activated by tumour)
Responders R (activated by tumour)
Expression of ~500 genes altered by >1.5X (P<0.05) in responding versus non-responding
NK cells
in
out out
in
CD107 accessible To Ab
0
10
20
30
40
50
60
70
SPR
Y2
HA
VC
R2
EGR
2
SPR
Y1
TRIM
5
IFN
G
CSF
2
Microarray R/NR
mR
NA
R/N
R
1
10
20
50
60
200
225
250 Pooled
Single
567
129
541
R/NR >1.5; P<0.02
Cell surface receptors Signal transduction Effectors
NK Receptors
& other cell
surface
molecules
KIRS (x9)
KLRC1
KLRC2
CD226
CRTAM
VSTM3
SLAMF7
HAVCR2
CD69
CD160
ICOS
PTPRC
SELL
Cytokine &
TNF SF
Receptors
IL12RB2
IL21R
IL4R
TNFRSF1B
TNFRSF4
TNFRSF9
TNFRSF7
Exocytosis
& cytotoxic
RAB27A
STX11
GZMB
Signalling
SPRY1
SPRY2
MAP3K8
MAP2K3
PTPN22
CBLB
PIK3C2B PIK3CA
PIK3R1
TRAF5
LAX1
CDC42
SH2D1B
SH2B1
Cytokines,
chemokines
& TNFSF
CSF2
IL3
IFNG
IL8
CCL3
XCL1
TNFSF2
TNFSF6
TNFSF14
TNFSF9
TNFSF15
ADAM17
Transcription
Factors
NFIL3
NFATC1
NFAT5
REL
MYC
EGR1
EGR2
EGR3
PRDM1
XBP1
Genes upregulated in tumour responding NK cells (~4hrs)
AU rich element
HVEM axis
TNFRSF14
(HVEM)
TNFSF14
(LIGHT)
TNFSF15
(TL1A)
TNFSF2
(TNFα)
CD160
TNFRSF1A
and TNFSF1B
NF-κB
TNFRSF6B
(DcR3)
TNFRSF25
(DR3)
TNFSF6
(FASL)
TNFRSF6
(Fas)
No target +K562
TN
FS
F1
4
R/NR~13
CD107
Tumour activated NK cells induce TNFSF14
(a pro-inflammatory cytokine)
What does TNFSF14 do?
• Transmembrane cytokine, expressed at surface and cleaved from surface
• Promotes anti-tumour CD8 responses
• Promotes memory T cell responses
• Aids influx of immune cells to secondary lymphoid tissue
• Activates endothelium (like TNF)
• Aids DC maturation (like TNF); is this a mechanism by which NK-DC cross-talk might occur?
NR
Fold
incre
ase C
D86
R R + αTNFSF14
5
10
15
20
25
+ cAb
1
* **
CD107
TN
FS
F14
Post-sort NK
NR
-NK
R
-NK
NK + K562
(4 hrs) +cAb
+cAb
+NR-NK
+cAb
+R-NK
+αTNFSF14
+R-NK
CD86
iDC
Pre-sort NK
CD
56
CD107
• TNFSF14 aka LIGHT
• Lymphotoxin-like, exhibits inducible expression, and competes with Herpes Simplex Virus glycoprotein D
for HVEM, a receptor expressed on T lymphocytes.
• Tumour activated NK cells make TNFSF14/LIGHT which aids DC maturation
• NK-DC crosstalk activates both the NK cell and the DC and important in initiating adaptive immunity
Holmes TD et al (2014) Licensed human natural killer cells aid DC maturation via TNFSF14/LIGHT
PNAS 111 (52): E5688-96.
How is TNFSF14 expression induced by tumour cells?
Tumour cell
NK cell
Which NK cell activation receptors signal
to induce degranulation and TNFSF14 production?
(use Ab to cross-link single/multiple receptors)
No Ab
14.4 3.9
1.6 CD107
TN
FS
F1
4
K562
19.9 24.7
3.7 Co
ntr
ols
NKp30 NKp44 NKp46
0.7 0
0
14.9 4.9
2.9
13.7 7.1
5
2B4
18.5 14.3
6.6
NKG2D
19.2 9.4
4.2
DNAM-1
18.1 7.8
4.2
CD16
39.1 33.6
5.3
NKp30
NKp44
NKp46
2B4
NKG2D
DNAM-1
18.9 46.2
11.5
NKp30
NKp44
NKp46
8.9 8.3
7.8
NKp46
2B4
NKG2D
DNAM-1
15.8 30.5
10.3
TNFSF14 and degranulation
• Induced by synergistic activity of multiple activation receptors
• The exception; CD16 cross-linking is sufficient
• True also for IFN-gamma, chemokines, TNF (other labs)
• IL-2 and IL-15 (not IFN-I) also induce TNFSF14 S
ing
le R
ece
pto
rs
Mu
ltip
le R
ece
pto
rs
NK cells are more than just killers
• They detect and kill targets, helping to slow the spread of infection or growth of a tumour
and
• Detection is coupled to release of cytokines (TNF, IFNg, GM-CSF and TNFSF14) plus chemokines that instruct adaptive immunity
• In particular, lack of PAMPs on tumour cells is overcome by altered-self recognition, cytokine production and DC activation.
Marvel Comics 1962
with a requirement for careful regulation; ”with great power comes great responsibility”
Production of pro-inflammatory cytokines in response to self-cells presents a potential problem
Why are there so many non-responders?
What safeguards against damage to healthy tissue?
19.1%
CD107
1.1%
CD
56
No target cells + tumour target cells (4hrs)
No
Yes
Problem 1
MHC class I
V. highly polymorphic
Chr 6
KIRs
Highly polymorphic
Chr19
Your KIRs might not be engaged by your MHC
Yes
Problem 2
KIRs are expressed clonally and stochastically
• Many NK cells express a single KIR
Its MHC ligand might not be expressed in the host
• A few NK cells express 2 or 3 KIRs
• A few NK cells express no KIRs
• These cells cannot detect MHC
Yes
Both scenarios have the potential to generate NK cells that are not inhibited by healthy tissue
These NK cells would kill healthy tissue and promote inflammation
The simplest example is the B2M -/- mouse
The B2M -/- mouse is viable, fertile and healthy,
but it has no cell surface MHC class I
Why don’t NK cells in B2M-/- mice attack healthy (MHC deficient) tissue?
(many cells express activation ligands when they divide)
Fresh NK cells from MHC class I deficient hosts (mouse and human)
are hyporesponsive to target cells
Potentially dangerous NK cell reactivity is disabled
How is NK cell tolerance achieved? NK cell education/licensing
During NK cell development:
The NK cell needs to engage with a cognate MHC class I using an inhibitory receptor
This engagement (which is ITIM dependent) confers ability to respond to target cells
• only NK cells that can recognise your self-MHC are licensed to kill
19.1%
CD107
CD
56
Responsive NK cells
Licensed
Educated
Express an inhibitory receptor against a self-MHC class I molecule
Non-responding NK cells
Hyporesponsive
Unlicensed Do not express an inhibitory receptor against a self MHC class I molecule
Uneducated Could be dangerous- no opportunity for inhibition by healthy cells
CD107
CD
56
Non-responders NR
(not activated by tumour)
Don’t make TNFSF14
Unlicensed?
No inhibitory receptors
against self-MHC?
Responders R
(activated by tumour)
Make TNFSF14
Licensed?
Express inhibitory receptors
against self-MHC? 0
500
1000
1500
2000
2500
0 KIR 1KIR 2KIR 0 KIR 1KIR 2KIR
NKG2A- NKG2A+
TN
FS
F1
4 (
MF
I)
* **
** *
*
NKG2Aneg
TNFSF14
Unstimulated
Stimulated, NKG2Aneg, KIRneg
Stimulated, NKG2A+, 2xKIR+
No inhibitory receptors for self-MHC class I
Three inhibitory receptors for self-MHC class I
TNFSF14 production is wired into the licensing mechanism (control of inflammation)
19.1%
CD107
CD
56
Responsive NK cells
Licensed/Educated
Non-responding NK cells
Hyporesponsive
Unlicensed/Uneducated
Useless?
• Cytokine stimulation allows the unlicensed cells to
respond (the same with degranulation and IFNg)
• Allows mobilisation of “uninhibitable” NK cells
when needed (calling up the army reserves….)
• Presumably limited by localised cytokine production
• Uninhibitable cells might cause collateral damage
• A potential source of tissue damage during infection or
inflammation?
media
IL-2
IL-15
media
IL-2
IL-15
0
200
400
600
800
1000
pg T
NF
SF
14 *
*
**
*
nsns
Cell sort licensed
and unlicensed cells (based on KIR expression)
Stimulate with IL-2 or IL-15
Licensed Unlicensed
Note: Not formally Licensed/unlicensed
Switching NK cells on and off
• On/off regulated by the balance of activating and inhibitory signals from an array of receptors
• Target cell MHC class I plays a key role in regulation of NK cell activity → regulation by missing self
• Ligands induced by cell “stress” (including infection and sustained proliferation) activate NK cells → regulation by altered self
• Activated NK cells kill targets and produce cytokines that aid innate and adaptive immunity
If NK cells are so good at detecting tumour cells, then why do we get cancer?
Compare NK cells in the tumour microenvironment (niche) versus periphery
Patient peripheral blood NK
Malignant ascites (drain) NK cells
Healthy donor peripheral blood NK
Tumour associated NK cells have
reduced expression of activation
receptors
Tumour-derived NK cells less active
Effect localised to tumour
Peripheral blood NK cells unaffected DNAM-1
P1
1. Ovarian Cancer:
2. Brain Cancer (Glioblastoma):
Patient peripheral blood NK
Brain tumour (surgery) NK
Healthy donor peripheral blood NK
Compare surface phenotypes
Functional studies (where possible)
How do tumours cell evade NK cells? (example; ovarian cancer)
Granzyme B
Actin
IL-15+ TGF-β
Inhibition of the cytotoxic apparatus
+15 15+β Unstim
NKG2D
Tumour-sensing activation
receptors (several)
Inhibition of proliferation
CSFE
Prolif
TGF-β From tumour cells
myeloid cells, Treg
NK cell survival
Survival is unaffected
NK cells are alive
but inhibited
Allows activity to be restored
via antibodies or
small molecule inhibitors
NK
p46
Post-culture stimulus
None K562
NK
alo
ne
Culture
NK
+ O
vT
um
or
+Ig
G1
NK
+ O
vT
um
or
+ a
nti-T
GF
-β
3%
IFN-γ
28.3%
5% 18%
6% 25.1%
Wholly autologous ex vivo model
Primary ovarian cancer cells
+ NK cells from same patient blood
NK cells adopt phenotype of those in the tumour
Reversible with TGF-B antagonists
T cell and NK cell surface phenotype in brain tumours (glioblastoma multiforme)
Immune checkpoints PD1 and LAG3 upregulated NK cell activation receptors NKp30, NKG2D
and DNAM-1 downregulated
T cells (bulk CD3+) NK cells (bulk NKp46+)
Weakened T cells Weakened NK cells
Mechanism? TGF-B?
28% 78%
CD279/PD1
Patient blood Patient tumour
CD226/DNAM-1
89% 49%
Can we exploit NK cells in cancer therapy?
1) Existing huMab therapy e.g Rituximab, Herceptin; ADCC
2) Agents in trials
e.g. anti-KIR huMab
Akin to checkpoint
blockade
Yes
3) Oncolytic viruses (e.g. Reovirus)
Act in two ways
i) Kill tumour cells directly
ii) Promote anti-tumour immunity
Clinical trials in Leeds allow
assessment of activity in vivo
1010 U virus
0hr blood
Cancer patient (n=10)
1hr blood
48hr blood
96hr blood
Surgery day (SD)
blood
Surgery
(1-4 weeks)
1 Mo blood
3 Mo blood
1010 U virus
1010 U virus
1010 U virus
1010 U virus
Iso
0hr
1hr ns
48hr
96hr
SD
1 Mo
3 Mo
CD69
Iso
0hr
24hr
72hr
SD
1 Mo
3 Mo
ns
ns
ns
1hr
18%
6%
63%
5%
3%
4%
4%
2%
2%
2%
3%
1%
2%
6%
6%
46%
44%
30%
6%
3%
7%
5%
35%
7%
HC P3 P8 P1
Total NK cells (n=10)
0
20
40
60
80
100
0 1 48 96 SD 1Mo 3Mo
CD
69
+ (
%)
ns- not sampled
• A single peak of NK cell activation co-incident with IFN-I response
(note that patients did not show response to later doses; Ab?, refractory to IFN?)
• In vitro experiments confirm that NK cell activation is IFN-I dependent
• Mouse models show that antitumour activity of Reo is NK cell dependent
Both CD56bright and CD56dim NK cells are activated by reovirus in vivo
100 101 102 103 104
FL1-H
p2 1h dnam -1-16.012
R4
CD
56
CD16
CD56brightCD16low/neg
make IFN-γ in response to IL-12/18 weak cytotoxic activity 10% in blood but 90% in LN (also express LN homing molecules)
CD56dimCD16+ strong cytotoxic activity 90% in blood but 10% in LN
NK differentiation
%C
D6
9+
%C
D6
9+
brights
dims
1hr 48hr 96hr S-day 30 days 0hr
10% 12% 3% 7% 7% 11%
0 1 48 96 SD 1Mo 3Mo 0
5
10
15
20
25 P=0.016
P=0.011
CD
56
brig
ht (
%)
CD56bright NK cells disappear from peripheral blood at peak of activation
Peripheral circulation Lymph node
dim
bright S1P S1PR
CD69
reovirus
CCR7
IFN-I
CD
69
(%
)
Peak of NK cell activation
Model for oncolytic virus action
IFN-I activates NK cells and;
i) Increases cytotoxicity towards tumour
ii) Alters trafficking; brights to LN where they make
IFN-g and favour cytotoxic T cell responses?
Summary
• NK cells are regulated by missing/altered self via a repertoire of activating and inhibitory receptors
• MHC class I regulates the response to individual target cells and the licensing of individual NK cells
• NK cells do more than their name suggests; they kill and they modulate the immune response
• (e.g. via cytokines and DC maturation)
• NK cells couple tumour immune surveillance to DC maturation and adaptive immunity
• They are inhibited in the tumour microenvironment, e.g. by immunosuppressive cytokines such as TGF-B
• They have proven clinical activity against cancer and defining the pathways via which they are inhibited by tumours
and understanding how different agents modulate NK cell responses will enhance future cancer therapy.
Thanks to:
Tim Holmes (now in Bergen)
Erica Wilson
Helen Close (now in Oxford)
Laura Wetherill
Emma Black
Yasser El-Sherbiny
Abbie Neilson (now making Whisky)
Michelle Wantoch
Sian Drake
Sarah Phillips
Aarren Mannion
Adam Odell
Vicky Jennings
Josie Meade
Tony Bramall
Scholarships
Alan Melcher (now at ICR, London)
Susan Short
Eric Blair
Vivek Tanavde (A-Star Singapore)
