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.