Natural Killer Cells in Chronic Hepatitis B and Chronic Hepatitis C Virus Infections HBV and HCV infections differ in their viral replication cycles, kinetics of infection, and natural history of disease progression. NK cells play an important role in controlling HBV and HCV infections. In chronic HBV and HCV infections, NK cells exhibit functional defects including reduced cytokine production and cytotoxicity. Exposure to HCV can directly impair NK cell degranulation and TRAIL expression. Antiviral therapies may help restore NK cell function in chronic viral hepatitis.

1. Natural Killer Cells in Chronic
Hepatitis B and Chronic
Hepatitis C Virus Infections
Mario U. Mondelli
Research Laboratories, Department of Infectious
Diseases, Fondazione IRCCS Policlinico San Matteo and
Department of Internal Medicine, University of Pavia, Italy.

2. HBV and HCV
Broad Differences in Viral Replication

3. Broad Differences in HBV and HCV Replication
HBV1,2
Host cell
Host cell
cccDNA
Host DNA
HCV1,3
H
Host DNA
HCV RNA
H
Integrated DNA
Nucleus
Long-term suppression
of viral replication
Nucleus
Definitive viral clearance
and SVR
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.

4. Kinetics of HBV and
HCV Replication
100
HCV
10
HBV
1
8
18
2
10
4
12
6
14
16
Weeks after infection
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%

5. HBV and HCV
Broad Differences in Natural History

6. (Slow)
Young age at infection
 30 years
Progression
HCC
(1-4%/yr)
Normal
Liver
Acute
Infection
Chronic
Hepatitis
Cirrhosis
(20 %)
Decompensation
IL-28B Polymorphism
(Fast)
Chronic
Infection
(60-80%)
 20 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.

7. immune
tolerance
<
immune
clearance
HBeAg + (wild)
low or non
replicative phase
><
reactivation
phase
HBeAg - / anti-HBe +
>
>105 cp/ml
HBV-DNA
109-1010 cp/ml
<105 cp/ml
107-108 cp/ml
ALT
mild CH
moderate/severe CH
HAI < 4
cirrhosis
HBeAg + CH
moderate/severe CH
cirrhosis
inactive-carrier state
resolved hepatitis B
(HBsAg - / anti-HBs +)
HBeAg - CH

8. HBV and HCV
Are NK Cells Important in Infection
Control ?

9. NK Cell Functions
CD56bright/CD16(10%)
Cytokine secretion
(TNF-, IFN-, TGF-, GM-CSF)
FL3H: CD56 G2b PE-Cy5
CD56 PE-Cy5
10
4
10
3
10.3
80.2
CD56dim/CD16+/(90%)
10
2
Cytotoxicity vs virus-infected
or tumour targets
101
6.78
100
100
2.65
101
102
103
FL4-H: CD16 u APC
CD16 (FcRIII) APC
104

10. NK-Cell Function Is Regulated by Signalling Through
Activating and Inhibitory Receptors.

11. Increased Production of IFN by NK Cells in
Acute HCV Infection Regardless of Clinical Outcome
Amadei, et al. Gastroenterology. 2010;138:1536-45.

12. Increased NK Cell Activating Receptor Expression
in Acute vs Chronic HBV Infection
Zhao, PLoS ONE 2012;7:e49135

13. NK Cell Functional Dichotomy in Patients with
Chronic HCV Infection
Phenotype skewed towards activation
NKG2D
100
%
KIR3DL1
30
%
p=0.012
75
p=0.0050
Dysfunctional cytokine production
% 100
50
TNF
80
60
20
IFN
p=0.041
p=0.009
40
10
25
20
0
0
HCV
CTRL
CTRL
HCV
0
CTRL
HCV
CTRL
HCV
Increased cytotoxic activity
%CD107a NK cells
60
Media
IL2+IL12
IL2+IL21
50
p=0.003
40
p=0.001
30
p=0.010
20
10
0
CTRL
HCV
CTRL
HCV
CTRL HCV
Oliviero et al Gastroenterology 2009;137:1151-60.
Oliviero et al Gastroenterology 2009;137:1151-60.

14. 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

15. NK Cell Functional Dichotomy in Chronic HCV Infection
IFN Polarizes NK Function Towards Cytotoxicity and Reduced IFN Secretion
Mondelli MU, et al. Front Immunol 2012;3:351.

16. Dysfunctional NK Cytotoxicity and Cytokine Production
in Chronic HBV Infection
100
% CD107a+ NK cells
NS
75
IL2+IL12
IL2+IL21
p=0.009
p=0.018
50
25
0
BC
IL2+IL12
HD HBV
HD HBV
80
IL2+IL21
60
p=0.013
40
p=0.004
20
0
%TNF+ NK cells
% IFN+ NK cells
80
HBV
IL2+IL12
IL2+IL21
60
p=0.023
40
p=0.002
20
0
HD
HBV
HD
Oliviero et al. Gastroenterology 2009;137:1151-60
HBV
HD
HBV
HD
HBV

17. IL-10 Is Elevated in CHB and Suppresses
NK Cell IFN Production
Peppa et al., PLoS Pathog. 2010; 6: e1001227

18. IL10/TGF Blockade Enhances Intrahepatic
NK Cell IFN Production
Peppa et al., PLoS Pathog. 2010; 6: e1001227

19. Deficient NK Cell Function in Immunotolerant Patients
with Chronic HBV Infection: Effect of Antiviral Treatment
Sun et al.. PLoS Pathog 2012;8:e1002594

20. HBV and HCV
How Do NK Cells Behave in the Liver ?

21. Relative Frequencies of PB and Liver
Lymphocyte Subsets
BLOOD
B
LIVER
T
NK
B
NK
13%
T 10% 10%
13% NKT
3%
2%
NKT
T
6%
72%
Modified from O’Farrelly Immunol Rev 2000.
NK
31%
37%
72%
T
T
B
6%
20%
T
T
NKT
NKT

22. The Proportion of Intrahepatic NK Cells Is
Reduced in Chronic HCV Infection
40
p<0.0001
20
10
% IH NK cells
% IH NK cells
30
20
10
0
0
CTRL
HCV
Varchetta et al, Hepatology 2012;56:841-9
p=0.008
30
1-7
8-12
HAI score

23. Increased NCR and NKG2D Expression and Downregulation
of TRAIL and CD107a in Chronic HCV Infection
NKp30
NKG2D
NKp46
20
MFI
MFI
10
p=0.031
100
40
30
20
50
5
p=0.043
50
MFI
p=0.08
15
60
150
10
0
0
0
CTRL
CTRL
HCV
CTRL
HCV
TRAIL
10.0
15
p=0.012
7.5
p=0.034
10
MFI
% pos. NK cells
CD107a
5
5.0
2.5
0.0
0
CTRL
Varchetta et al, Hepatology 2012;56:841-9
HCV
CTRL
HCV
HCV

24. p=0.03
75
50
25
0
% CD107a+ NK cells
CTRL
n=10
HCV
n=11
P815 + anti NKG2D
75
p=0.03
50
25
0
CTRL
n=11
Varchetta et al, Hepatology 2012;56:841-9
HCV
n=13
% CD107a+ NK cells
K562
100
% CD107a+ NK cells
% CD107a+ NK cells
IH NK Cell Cytolytic Potential Is Impaired in Patients
with Chronic HCV Infection
P815
75
p=0.02
50
25
0
CTRL
n=13
HCV
n=14
P815 + anti NKp30
75
p=0.01
50
25
0
CTRL
n=10
HCV
n=10

25. CHRONIC HCV
INFECTION
NKR ligands
Continuous NK receptor
engagement and modulation
Degranulation
NK
Unresponsiveness?
Inhibitory
Cytokine(s),
e.g. IL-10 ?
TGF ?
Exhaustion?
↓ TRAIL
↓ CD107a

26. Higher Expression of Activation Markers and Degranulation in
Chronic Hepatitis Compared with Immunotolerance to HBV
Zhang et al. Hepatology 2011;53:73-85

27. HBV and HCV
Can Viruses Influence NK Cell Phenotype
and Function ?

28. HCV Core1 and NS32 Staining of Huh-7.5 Cells3
Core
NS3
FITC
Uninfected
Infected
Infected (caption)
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.

29. Exposure to HCVcc Impairs NK cell Degranulation and Ability to Upregulate
TRAIL in HCV patients
6
6
HCV-RNA: 0
3.64
4
TRAIL
2.5x10 cp/ml
2.97
4
3
10
3
3
10
50
40
30
10
10
102
102
1
10
1
10
0
0
10
0
10
96.4
1
10
2
10
0
0
10
10
3
4
10
0
10
10
1
10
2
10
3
0
20.2
3
2
10
45.2
10
0
10
0
10
1
10
2
10
3
0
10
0
4
10
10
10
CTRL
1
10
100
2.5x106 15x106
2
1
1
1
10
2
10
3
10
4
10
0
1
10
2
10
3
10
4
10
10
CD56+CD3-
HCV-RNA cp/ml
CD107a
HCV-RNA: 0
53.6
4
10
2.5x106cp/ml
45.2
4
15x106cp/ml
47.1
4
10
10
10
3
10
3
10
3
2
10
2
10
1
10
1
10
90
10
80
CD107a
% CD107a+ NK cells
10
10
10
0
4
10
10
2
2.5x106 15x106
3
10
3
10
10
0
2
10
10
3
10
1
10
10
4
10
2.67
0
4
10
4
4
10
HCV
1
10
10
20
15x10 cp/ml
13.4
4
10
102
60
TRAIL
% TRAIL+ NK cells
10
70
60
10
0
1
0
10
0
10
0
10
1
10
2
10
3
10
4
4
10
0
10
10
1
10
2
10
3
10
4
10
1
10
2
10
3
4
10
10
4
10
36.6
0
10
4
10
HCV
2
10
65.8
10
3
10
3
2
10
2
10
1
10
1
77.9
10
3
10
50
10
40
30
10
0
0
2.5x106 15 x106
0
2.5x10615 x106
HCV-RNA cp/ml
Varchetta et al, Hepatology 2012;56:841-9
1
0
10
1
10
2
10
3
10
4
10
1
0
10
0
10
CTRL
2
10
0
10
1
10
2
10
3
10
4
10
CD56+CD3-
0
10
1
10
2
10
3
10
4
10

30. 6
%CD56+CD3-pERK+
CONTROLS
5
4
3
2
1
0
T0
T15
T5
HCV+ PATIENTS
5
4
3
2
1
0
T30
0.91
2.92
2
10
101
1
10
100
100
101
102
103
104
104
0
10
0
10
1
10
2
10
3
10
4
10
104
3.68
103
6.04
103
HCV POS
MEDIUM
102
102
101
101
100
101
102
103
104
100
0.43
101
anti-pERK1/2
102
103
104
0.52
3
10
2
10
1
10
HCV NEG
MEDIUM
2
10
1
10
0
10
0
10
1
10
2
10
3
10
4
10
4
10
0
10
0
10
1
10
2
10
3
10
4
10
4
10
3
10
3
10
0.12
2
10
0.04
HCV POS
MEDIUM
2
10
1
10
100
100
4
10
3
10
HCV NEG
MEDIUM
T30
T30’
4
10
3
10
102
T15
T15’
4
10
103
T5
T0
T30’
T15’
104
CD56+ CD3-
6
CD56+ CD3-
%CD56+CD3-pERK+
Decreased ERK1/2 Phosphorylation in HCVcc-Exposed NK Cells from HCV Patients
1
10
0
10
0
10
1
10
2
10
3
10
4
10
0
10
0
10
1
10
2
10
3
10
4
10
anti-pERK1/2
PBMC+HCV NEG MEDIUM
Varchetta et al, Hepatology 2012;56:841-9
PBMC+HCV POS MEDIUM

31. IFN Rescues TRAIL Expression on NK Cells from HCV-Infected Patients
HCV-RNA: 0
15*106 cp/ml
HCV-RNA: 0
6.2
15*106 cp/ml
4.9
3.8
31.8
TRAIL
- IFN
57.4
43
17.4
25.1
+ IFN
HD
CD56+/CD3-
HCV

32. IFN Rescues NK Cell Degranulation in HCV-Infected Patients
16.4
15*106 cp/ml
HCV-RNA: 0
15*106 cp/ml
HCV-RNA: 0
32.8
34
34.6
CD 107a +
- IFN
45.1
39.9
43.4
46.1
+ IFN
HD
CD56+/CD3-
HCV

33. HBV Inhibits Cytokine Production and
pDC-Induced NK Cell Activation
Woltman, PLoS ONE 2011;6:e15324.

34. HBV and HCV
What is the Effect of IFN-Based
Therapies on NK Cells?

35. Baseline NK cell Phenotype in Patients with Different
Treatment Outcomes
%NK CD56dim
Cytotoxicity
Cytokine
production
Perforin Content
0.02
0.02
bright
0.04
CD56
dim
HD
SVR
NR
REL
HD
SVR
NR
REL
NKp30 expression
0.0002
0.049
CD3
0.003
Healthy Donors (HD)
Sustained Virologic Responders
(SVR)
Non-Responders (NR)
Relapsers (REL)
HD
SVR
NR
REL
Oliviero, EASL 2012

36. Higher Frequency of Activated NK Cells in RVR and SVR
Patients with Chronic HCV Treated with PEG-IFN + RBV
%CD69+ NK cells
30
***
***
20
*
10
SVR
*
NR
RVR
0
0
2
4
12
Time (weeks)
Oliviero, submitted
*p≤0.05, **p≤0.01, ***p≤0.001

37. Immunological Profile of SVR vs NR Patients Defined by
Linear Regression Analysis for Repeated Measurements
Higher CD16 (FcRIII) expression
p=0.017
Perforin/Isotype MFI
NR
CD16 MFI
SVR
0
12
NR
SVR
0
CD16
CD56
NR
2
4
Time (weeks)
SVR
Perforin
SVR
2
4
Time (weeks)
Lower Perforin content
p=0.014
CD56
NR
12

38. Cytolytic Potential (Degranulation)
IL2 + IL12
0.02
0.03
0
2
4
Time (weeks)
SVR
NR
Oliviero, submitted
0.002
%CD107a+ NK cells
%CD107a+ NK cells
IL2 + IL21
12
0.002
0
0.03
2
4
Time (weeks)
12

39. Impact of PegIFN on NK Cell Effector Function in CHB
Micco J Hepatol 2012, in press

40. Acknowledgements
Research Laboratories, Dept. of Infectious Diseases, Fondazione IRCCS
Policlinico San Matteo and Dept. of Internal Medicine, University of Pavia
Lab Team (Innate Immunity):
• Stefania Varchetta
• Barbara Oliviero
• Stefania Mantovani
• Dalila Mele
• Chiara Gazzabin
Clinical Team:
• Serena Ludovisi
•Giuseppe Michelone
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.