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Natural Killer Cells in Chronic Viral Hepatitis
Citation preview
Natural Killer Cells in Chronic
Hepatitis B and Chronic
Hepatitis C Virus Infections
Mario U. Mondelli
Research Laboratories, Department of InfectiousDiseases, Fondazione IRCCS Policlinico San Matteo and Department of Internal Medicine, University of Pavia, Italy.
HBV and HCV
Broad Differences in Viral Replication
Broad Differences in HBV and HCV Replication
H
HBV1,2
Host cell
cccDNA
Host DNA
Integrated DNA
Nucleus
H
HCV1,3
Host cell
Host DNA
Nucleus
HCV RNA
Definitive viral clearance
and SVRLong-term suppression
of viral replication
Adapted from 1. Soriano V, et al. J Antimicrob Chemother 2008;62:1-4. 2. Locarnini S and Zoulim F. Antiviral Therapy 2010;15 (suppl 3):3-14.
3. Sarrazin C and Zeuzem S. Gastroenterology 2010;138:447-462.
Kinetics of HBV and HCV Replication
Weeks after infection
HBV1
10
100
2 4 6 8 10 12 14 16 18
HCV
Thimme, R. et al, J Exp Med 194, 1395-406 (2001)
Webster, G. J. M. et al, Hepatology 32, 1117-24 (2000)
HCV: persistence in 70%
HBV: persistence in <5%
HBV and HCV
Broad Differences in Natural History
Young age at infection(F
ast
)(S
low
)Pro
gre
ssio
n
Normal
Liver
Acute
Infection
Chronic
Infection
(60-80%)
Chronic
Hepatitis
Cirrhosis
(20 %)
20 years
30 years
Alcohol, steatosis and/or IR, coinfections (HIV, HBV), old age, male gender
Modified by Lauer et al., N Engl J Med 2001;345:41-52.
IL-28B Polymorphism
HCC
(1-4%/yr)
Decompensation
< <> >HBeAg + (wild) HBeAg - / anti-HBe +
ALT
HBV-DNA
mild CH moderate/severe CH moderate/severe CHHAI < 4
cirrhosis
inactive-carrier state
resolved hepatitis B
(HBsAg - / anti-HBs +)
HBeAg - CHHBeAg + CH
immune
tolerance
immune
clearance
low or non
replicative phase
reactivation
phase
cirrhosis
109-1010 cp/ml107-108 cp/ml
<105 cp/ml
>105 cp/ml
HBV and HCV
Are NK Cells Important in Infection Control ?
NK Cell Functions
100
101
102
103
104
FL4-H: CD16 u APC
100
101
102
103
104
FL
3H
: C
D5
6 G
2b
PE
-Cy
5
10.3 80.2
2.656.78
CD16 (FcRIII) APC
CD
56 P
E-C
y5
CD56dim/CD16+/-
(90%)
CD56bright/CD16-
(10%)
Cytotoxicity vs virus-infected
or tumour targets
Cytokine secretion
(TNF-, IFN-, TGF-, GM-CSF)
NK-Cell Function Is Regulated by Signalling Through
Activating and Inhibitory Receptors.
Increased Production of IFN by NK Cells in
Acute HCV Infection Regardless of Clinical Outcome
Amadei, et al. Gastroenterology. 2010;138:1536-45.
Increased NK Cell Activating Receptor Expression
in Acute vs Chronic HBV Infection
Zhao, PLoS ONE 2012;7:e49135
NK Cell Functional Dichotomy in Patients with
Chronic HCV Infection
Oliviero et al Gastroenterology 2009;137:1151-60.
Increased cytotoxic activity
p=0.003p=0.001
%C
D1
07a
NK
ce
lls
IL2+IL12 IL2+IL21Media
0
10
20
30
40
50
60
p=0.010
CTRL HCV CTRL HCVCTRL HCV
Dysfunctional cytokine production
p=0.041 p=0.009
IFN TNF
CTRL HCV CTRL HCV0
20
40
60
80
100%
%
Phenotype skewed towards activation
NKG2D
CTRL HCV 0
25
50
75
100p=0.012
KIR3DL1
CTRL0
10
20
30p=0.0050%
HCV
Oliviero et al Gastroenterology 2009;137:1151-60.
PegIFN/RBV Therapy Results in Preferential Phosphorylation of STAT1 over STAT4 in NK Cells from HCV-Infected Persons
Edlich et al. Hepatology 2012;55:39-48
Mondelli MU, et al. Front Immunol 2012;3:351.
NK Cell Functional Dichotomy in Chronic HCV InfectionIFN Polarizes NK Function Towards Cytotoxicity and Reduced IFN Secretion
HD HBV HD HBV
0
20
40
60
80 IL2+IL12 IL2+IL21
p=0.013
p=0.004
% IF
N+
NK
ce
lls
HD HBV HD HBV
0
20
40
60
80 IL2+IL12 IL2+IL21
p=0.023
p=0.002%
TNF
+ N
K c
ells
BC HBV HD HBV HD HBV
0
25
50
75
100NS IL2+IL12 IL2+IL21
p=0.009 p=0.018
Oliviero et al. Gastroenterology 2009;137:1151-60
% C
D1
07
a+ N
K c
ells
Dysfunctional NK Cytotoxicity and Cytokine Production
in Chronic HBV Infection
IL-10 Is Elevated in CHB and Suppresses
NK Cell IFN Production
Peppa et al., PLoS Pathog. 2010; 6: e1001227
Peppa et al., PLoS Pathog. 2010; 6: e1001227
IL10/TGF Blockade Enhances Intrahepatic
NK Cell IFN Production
Deficient NK Cell Function in Immunotolerant Patients
with Chronic HBV Infection: Effect of Antiviral Treatment
Sun et al.. PLoS Pathog 2012;8:e1002594
HBV and HCV
How Do NK Cells Behave in the Liver ?
Modified from O’Farrelly Immunol Rev 2000.
NK
13%
T
72%
NKT
B T
10%
BLOOD
T
72%
T3%
B
10%NK
NKT
13%2%
NKT
LIVER
T
T
37%
T6%
B
6%NK
NKT
31%
20%
Relative Frequencies of PB and Liver
Lymphocyte Subsets
CTRL HCV
0
10
20
30
p<0.0001
% I
H N
K c
ell
s
% I
H N
K c
ells
HAI score
p=0.008
1-7 8-120
10
20
30
40
The Proportion of Intrahepatic NK Cells Is
Reduced in Chronic HCV Infection
Varchetta et al, Hepatology 2012;56:841-9
10.0
TRAIL
CTRL HCV
0.0
2.5
5.0
7.5 p=0.012
NKp30
MF
I
MF
I
MF
I
NKp46
MF
I
0
10
20
30
40
50
60
HCVCTRL
p=0.043
NKG2D
0
50
100
150
HCVCTRL
p=0.031
CTRL HCV
0
5
10
15
20
p=0.08
Increased NCR and NKG2D Expression and Downregulation
of TRAIL and CD107a in Chronic HCV Infection
CTRL
% p
os. N
K c
ell
s
p=0.034
0
5
10
15
CD107a
HCV
Varchetta et al, Hepatology 2012;56:841-9
% C
D107a
+ N
K c
ell
s
CTRL HCV
P815 + anti NKp30
0
25
50
75
p=0.01
% C
D107a
+ N
K c
ell
s
0
25
50
75
p=0.03
CTRL HCV
P815 + anti NKG2D
CTRL HCV
P815
0
25
50
75
p=0.02
% C
D107a
+ N
K c
ell
s
% C
D1
07
a+
NK
ce
lls
CTRL HCV0
25
50
75
100
p=0.03
K562
n=11n=10 n=14n=13
n=13n=11 n=10n=10
IH NK Cell Cytolytic Potential Is Impaired in Patients
with Chronic HCV Infection
Varchetta et al, Hepatology 2012;56:841-9
CHRONIC HCV
INFECTION
Unresponsiveness?
Degranulation
Exhaustion?
Inhibitory
Cytokine(s),
e.g. IL-10 ?
TGF ?
NKR ligands
Continuous NK receptor
engagement and modulation
NK
↓ TRAIL
↓ CD107a
Higher Expression of Activation Markers and Degranulation in
Chronic Hepatitis Compared with Immunotolerance to HBV
Zhang et al. Hepatology 2011;53:73-85
HBV and HCV
Can Viruses Influence NK Cell Phenotypeand Function ?
FITC
Core NS3
Uninfected Infected Infected (caption)
HCV Core1 and NS32 Staining of Huh-7.5 Cells3
1. Cerino A, et al. J Immunol 1993;151:7005-15; 2. Mondelli MU, et al. J Virol 1994;68:4829-36; 3. Varchetta et al, Hepatology 2012;56:841-9.
CD107a
HCV-RNA cp/ml
0 2.5x106 15 x106 0 2.5x10615 x10630
40
50
60
70
80
90
100
101
102
103
104
100
101
102
103
104
100
101
102
103
104
100
101
102
103
104
100
101
102
103
104
100
101
102
103
104
100
101
102
103
104
100
101
102
103
104
100
101
102
103
104
100
101
102
103
104
100
101
102
103
104
100
101
102
103
104
1
CD56+CD3-
2.5x106cp/ml 15x106cp/mlHCV-RNA: 0
TRAIL
0 2.5x106 15x106 0 2.5x106 15x1060
10
20
30
40
50
60
HCV-RNA cp/ml
HCV
CTRL
HCV
CTRL
2.5x106 cp/ml 15x106cp/mlHCV-RNA: 0
CD56+CD3-
100
101
102
103
104
100
101
102
103
104
96.40
100
101
102
103
104
100
101
102
103
104
100
101
102
103
104
100
101
102
103
104
100
101
102
103
104
100
101
102
103
104
100
101
102
103
104
100
101
102
103
104
100
101
102
103
104
100
101
102
103
104
77.965.836.6
53.6 45.2 47.1
2.67 20.2 45.2
13.42.973.64
TR
AIL
% C
D1
07
a+
NK
cells
CD
10
7a
% T
RA
IL+
NK
ce
lls
Exposure to HCVcc Impairs NK cell Degranulation and Ability to Upregulate
TRAIL in HCV patients
Varchetta et al, Hepatology 2012;56:841-9
100 101 102 103 104100
101
102
103
104
100 101 102 103 104100
101
102
103
104
CONTROLS
T0 T5 T15 T300
1
2
3
4
5
6
%C
D56+
CD
3-p
ER
K+
PBMC+HCV NEG MEDIUM
PBMC+HCV POS MEDIUM
T15’ T30’
anti-pERK1/2
CD
56
+ C
D3
-
HCV NEG
MEDIUM
HCV POS
MEDIUM
0
1
2
3
4
5
6
T0 T5 T15 T30
%C
D56+
CD
3-p
ER
K+
anti-pERK1/2
T15’ T30’
CD
56
+ C
D3
-
HCV NEG
MEDIUM
HCV POS
MEDIUM
HCV+ PATIENTS
100 101 102 103 104
100
101
102
103
104
0.040.12
100 101 102 103 104100
101
102
103
104
0.43
100 101 102 103 104100
101
102
103
104
100 101 102 103 104100
101
102
103
104
2.92
100 101 102 103 104
100
101
102
103
104
0.91
100 101 102 103 104
100
101
102
103
104
3.68 6.04
0.52
Decreased ERK1/2 Phosphorylation in HCVcc-Exposed NK Cells from HCV Patients
Varchetta et al, Hepatology 2012;56:841-9
CD56+/CD3-
TR
AIL
HCV-RNA: 0 15*106 cp/ml
6.2 31.8
57.443
3.8 4.9
17.4 25.1
HCV
HCV-RNA: 0 15*106 cp/ml
+ IFN
- IFN
HD
IFN Rescues TRAIL Expression on NK Cells from HCV-Infected Patients
16.4
39.9
34
45.1
32.8
43.4
34.6
46.1
CD
107a +
CD56+/CD3-HD HCV
HCV-RNA: 0 15*106 cp/ml HCV-RNA: 0 15*106 cp/ml
+ IFN
- IFN
IFN Rescues NK Cell Degranulation in HCV-Infected Patients
Woltman, PLoS ONE 2011;6:e15324.
HBV Inhibits Cytokine Production and
pDC-Induced NK Cell Activation
HBV and HCV
What is the Effect of IFN-BasedTherapies on NK Cells?
NKp30 expression
HD SVR NR REL
0.0002
0.049
0.003
Baseline NK cell Phenotype in Patients with Different
Treatment Outcomes
%NK CD56dim
HD SVR NR REL
0.02
Perforin Content
HD SVR NR REL
0.02 0.04
CD
56
CD3
dim
bright
Healthy Donors (HD)
Sustained Virologic Responders
(SVR)
Non-Responders (NR)
Relapsers (REL)
Cytokineproduction
Cytotoxicity
Oliviero, EASL 2012
0 2 4 12
0
10
20
30
******
*
*
%C
D6
9+
NK
ce
lls
Time (weeks)
*p≤0.05, **p≤0.01, ***p≤0.001Oliviero, submitted
Higher Frequency of Activated NK Cells in RVR and SVR
Patients with Chronic HCV Treated with PEG-IFN + RBV
SVR
NR
RVR
0 2 4 12 0 2 4 12
Immunological Profile of SVR vs NR Patients Defined by
Linear Regression Analysis for Repeated Measurements
Higher CD16 (FcRIII) expression
p=0.017Lower Perforin content
p=0.014
SVR NR
CD56
SVR NR
CD56
Time (weeks) Time (weeks)
CD
16
Pe
rfo
rin
SVR NR SVR NR
CD
16
MF
I
Pe
rfo
rin
/Is
oty
pe
MF
I
0 2 4 12
0.002
0.0020.03
0 2 4 12
0.02
0.03
IL2 + IL21 IL2 + IL12
SVR
NR
Time (weeks) Time (weeks)
%C
D1
07
a+
NK
cells
%C
D1
07
a+
NK
cells
Cytolytic Potential (Degranulation)
Oliviero, submitted
Impact of PegIFN on NK Cell Effector Function in CHB
Micco J Hepatol 2012, in press
Acknowledgements
Lab Team (Innate Immunity):• Stefania Varchetta• Barbara Oliviero• Stefania Mantovani• Dalila Mele• Chiara Gazzabin
Clinical Team:• Serena Ludovisi•Giuseppe Michelone
Research Laboratories, Dept. of Infectious Diseases, Fondazione IRCCS Policlinico San Matteo and Dept. of Internal Medicine, University of Pavia
Collaborators:• Domenico Mavilio, Clinical & Experimental Immunology Lab, ICH.• Marco Montorsi, Riccardo Rosati, Department of Surgery, ICH.• Alessio Aghemo, Maria Grazia Rumi, Massimo Colombo, Division of Gastroenterology, Dept. of Medicine, IRCCS Ospedale Maggiore, Milan.