how does hbv interact with the immune...

Department of Medicine II, Medical Center - University of Freiburg, Germany

Robert Thimme

How does HBV interact with the immune system?

Course of HBV

Acute HBV

Chronic hepatitis B

95%

Elimination

5%

Persistence

Components of immune responses against HBV

Dendritic cells

NK cells

CD4+ T cells

Immunoregulatorycytokines

IL10

TGFβ

Innate immunityIFNα

CD8+ T cells

Hepatocytes

Treg

CD8+ T-cell responses in HBV infection

CD8+

weeks after infection

HBV DNA

T-ce

ll re

spon

se

0 2 4 6 8 10 12 14 16 18 20 22

T-cells

Depletion of CD8+ T cells prolongs viremia

Thimme, Chisari, J Virol 2003

weeks after infection

HBV DNA

T-ce

ll re

spon

se

Maini, Bertoletti Gastro 2001Boettler, Thimme, J Virol 2005

0 2 4 6 8 10 12 14 16 18 20 22

Temporal association of CD8+ T-cell responseand viral load

Intrahepatic accumulation of CD8+ T-cellresponses during viral clearance

Thimme, Chisari, J Virol 2003

viral load CD8 & AST

chronic phase

Thimme et al. J. Virol. 2003

cytolytic mechanisms

Function of CD8+ T cells in HBV

HBV infected hepatoma cellCD8+ T cell

Untreated control Direct co-culture Transwell cultures

- + stimulation

core18-27 peptide

Identifcation of a novel immunological cell culturemodel

Reduction of viral loads requires cell killing

Hoh et al. J Virol 2015

HBV

DNA

AST [U/L]

10 8

10 9

10 1 0

10 1 1

0

15

30

45

60

75

cytoplasmic HBV DNAAST

*

Δt = 96 hE:T = 1:1

untreated direct

Reduction of viral loads requires cell killing

Hoh et al. J Virol 2015

rel.

HBV

DNA AST [U/L]

10 8

10 9

10 1 0

10 1 1

0

15

30

45

60

75

cytoplasmic HBV DNAAST

*

Δt = 96 hE:T = 1:1

Strong reduction (95%) of viral loads requires:

• Direct cell-cell contact• Cytotoxic effector mechanisms

% re

mai

ning

vira

l DNA

0

20

40

60

80

100

p < 0.001

p < 0.05

HBeA

g PE

IU/m

l

0

10

20

30

40

50

HBeAg

HBsA

g [IU

/ml]

0

5

10

15

HBsAg

n = 2

Function of CD8+ T cells in HBV

GranzymePerforin

IFN-γTNF-α

cytolytic

non cytolytic

CD8+ H

CD8+ T-cell failureImmune responses in chronic HBV infection

priming

Treg cells

CD4+ T cells

immunoregulatorycytokines

IL10

TGFβ

TCR

antigen

CD8+ T cells exhaustion

viral escape

NK cells

Fate of virus-specific CD8+ T cells in acute andchronic infections

Acute:

Chronic:Exhausted:short-livedantigen-dependentfunctionally exhausted

Memory:long-livedantigen-independentpolyfunctional

Effector:short-livedantigen-dependentpolyfunctional

Heterogeneous populations -lessons from mouse model:• TbetdimEomeshi / TbethiEomesdim

(Paley, Science 2012)

• TCF1+ memory-like(Utzschneider, Nature Immunology 2013)

• CXCR5+ progenitor(Im & He, Nature 2016)

Aim of Study

Heterogeneity of virus-specific CD8+ T-cell populations in humans: chronic HBV and HCV infection

Inhibitory Receptors ↑↑↑• PD1• 2B4• TIGIT• KLRG1

Proliferation ↓↓↓

Cytokines↓↓↓ CD39 ↑↑↑

CD127 ↓↓↓(IL-7 Receptor)

Eomes↑↑Tbet ↓TCF1↓

Patient cohorts

cHBV (n=12):

• 7 inactive carriers− treatment naive

− low viral load: 779 IU/ml

(136 – 2149)

− no cirrhosis

• 5 patients with NUC therapy− low viral load: 19 IU/ml

(<10 – 33)

− no cirrhosis

cHCV (n=8):

• HCV genotype 1

• Viral load: 3213771 IU/ml

(42241 – 15x106)

• no cirrhosis

Alanio et al., Blood 2010Schmidt et al., J Virol 2011Nitschke et al., J Virol 2015

+PBMCs

epitope-specifictetramer

fluorochrome

magnetic anti-fluorochrome

microbeadlabeled PBMCs

PBMCs with enrichedepitope-specific T cells

magnetic-activated cell separation column

flow cytometric analysis

Magnetic bead enrichment of virus-specific CD8+ T cellsEnhanced resolution and potential for in-depth analysis of rare T-cell populations

Alanio et al., Blood 2010Schmidt et al., J Virol 2011Nitschke et al., J Virol 2015

+PBMCs

epitope-specifictetramer

fluorochrome

magnetic anti-fluorochrome

microbeadlabeled PBMCs

PBMCs with enrichedepitope-specific T cells

magnetic-activated cell separation column

flow cytometric analysis

Magnetic bead enrichment of virus-specific CD8+ T cellsEnhanced resolution and potential for in-depth analysis of rare T-cell populations

virus-specificCD8+ T cells

Before enrichment After enrichment

MF

I P

D1

H B V H C V0

2 0 0 0

4 0 0 0

6 0 0 0* *

PD1

tetra

mer

PD1

HBVHCV

CD8+

%E

om

esh

igh

H B V H C V0

2 0

4 0

6 0

8 0

1 0 0 * *

%C

D3

9+

H B V H C V0

2 0

4 0

6 0

8 0

1 0 0 *

Eomeshigh CD39

tetra

mer

Eomes

tetra

mer

CD39

Eomes CD39PD1

HBV: inactive carriers

HBV: patients withNUC therapy

HCV

HBV-specific CD8+ T cells lack terminal exhaustion markers

CD

127

PD1

HCVCD8+

HCV44.5

55.5

%C

D1

27

+P

D1

+

of

tet+

CD

8+

T c

ells

H B V H C V0

2 0

4 0

6 0

8 0

1 0 0**

%C

D1

27

- PD

1+

of

tet+

CD

8+

T c

ells

H B V H C V0

2 0

4 0

6 0

8 0

1 0 0***

CD127+PD1+CD127-PD1+

CD

127PD1

HBV98.6

0.8

CD8+

HBV

HBV-specific CD8+ T cells are predominantly CD127+PD1+

CD127+PD1+subset dominates in HBV-specific CD8+ T-cell populations



HCV

terminallyexhausted

less differentiated„memory-like“

CD

127

PD1

CD127+PD1+

Eomeslow

CD39-

TCF1high

CD127-PD1+

Eomeslow

CD39+

TCF1dim

Immunity 2016

Two subsets of exhausted virus-specific CD8+ T cells in HCV but not HBV

Fate of terminally exhausted CD8+ T cells after antigen elimination?

HBV

HCV

Baseline EOT

CD8+

HCV

CD

127

PD1

CD8+

HCV49

28 21

73

23 1

B a se line

E OT

F U1 2

0

2 0

4 0

6 0

8 0

1 0 0

%C

D1

27

- PD

1h

i

********

CD127/PD1 subsetsLess

differentiatedTerminallyexhausted

B ase line

E OT

F U1 2

0

2 0

4 0

6 0

8 0

1 0 0

%C

D1

27

+P

D1

+

******

C

DAA mediated antigen removal leads to disappearance of terminally exhausted HCV-specific CD8+ T cells

Dominik Wieland, in revision

0 2 0 4 0 6 0 8 0 1 0 00

1

2

3

% T C F 1 +

log

(E

xpa

ns

ion

In

de

x)

0.0006p =

0.4395r =

HCV



TCF1+

%T

CF

1+

of

tet+

CD

8+

T c

ells

H B V H C V0

2 0

4 0

6 0

8 0

1 0 0 **

Proliferative capacity(14d in vitro expansion)

The memory marker TCF1 determines proliferative capacity of virus-specific CD8+ T cells

High proliferative capacity of HBV-specific CD8+ T cellscorrelates with increased TCF1 expression

HCV



Bengsch / Thimme, J Hepatol 2014

PD-L1 blockade has the strongest effect

% n

orm

al. P

rolif

erat

ion

Nature 2016

T-cell subpopulations in chronicHBV and HCV infection

chronic HCV

Less differentiated/ memory-like

Terminal differentiated/

exhausted

chronic HBV

Determinant?

Lessdifferentiated/ memory-like

Terminal differentiated/

exhausted

Antigen recognition

CD39Eomes TCF1

VL: 30 405 IU/ml

CD

127

PD1

tetra

mer

CD39

tetra

mer

Eomes

tetra

mer

TCF1

HBVHCV

CD8+

Characteristics of T-cell subpopulations in chronicallyHBV-infected patients with high VL

Lack of terminally exhaustedHBV-specific CD8+ T cells isnot due to absent antigen

CD8+ T-cell failure

Immune responses to HBV

priming

Treg cells

CD4+ T cells

immunoregulatorycytokines

IL10

TGFβ

TCR

antigen

CD8+ T cells exhaustion

viral escape

NK cells

NK cells in chronic viral hepatitis

NK cell functional dichotomy in cHBVresponse to cytokine stimulation ↓↓

response to antibody-coated target cells ↑

HD cHBV

NK cell

cytokineresponsiveness ↓

IFNγ

cytokineresponsiveness ↑↑

IFNγ

ADCC ↑ADCC ↑↑

NK cell NK cellNK cell

NK cell

NK cell

NK cell

NK cellNK cell

NK cellNK cell

NK cellNK cell

NK cell

NK cellNK cell

NK cellNK cell

NK cell


Oliviero et al., Gastroenterology 2009

NK cells in chronic viral hepatitis

NK cell functional dichotomy in cHBV

Oliviero et al., Gastroenterology 2009Béziat et al., EJI 2012

response to cytokine stimulation ↓↓response to antibody-coated target cells ↑

Increased frequencies of NKG2C+ NK cells in chronic viral hepatitis→ linked to CMV seropositivity

HD cHBV

NK cell


NK cell

NK cell

NK cell

NK cellNK cell

NK cellNK cell

NK cellNK cell

NK cell

NKG2C ↑

NK cellNK cell

NK cellNK cell

NK cell


CMV


IFNγ


IFNγ

ADCC ↑ADCC ↑↑

Conventional vs. adaptive NK cells

adaptive NK cell

CD3ζ

NKG2C ↑

Helios ↓PLZF ↓

FcεRIγ

conventional NK cell

FcεRIγ

CD3ζHeliosPLZF

CD16CD16

Lee J et al., Immunity 2015Schlums H et al., Immunity 2015

ADCC ↑ADCC ↑↑

CMV

→ epigenetic regulation


IFNγ


IFNγ

Aim of Study

Is altered NK cell function in cHBV patients linked to an

expansion of adaptive NK cell subsets?

adaptive NK cell

conv.NK cell

adaptive NK cell

conv.NK cell

conv.NK cell

conv.NK cell

conv.NK cell

conv.NK cell

adaptive NK cell

adaptive NK cell

adaptive NK cell

adaptive NK cell

adaptive NK cell

HBV

Study approachMethods

Multiparametric flow cytometry• Phenotypic analyses• Functional analyses of cytokine

production and degranulation after − cytokine stimulation with IL-12/IL-15

and IL-12/IL-18− CD16 crosslinking (plate-bound)

Epigenetic analyses (EpigenDx)− FCER1G promoter methylation− IFNG promoter methylation

Patient cohorts

cHBV (n=21):

• no cirrhosis

• age: 41 (18 – 68)

• viral load: 8657512 IU/ml (0 – 1.64x108)

• ALT: 43 IU/ml (11 – 304)

• treatment: 16 naïve, 5 NUC

HD (n=30):• age: 42 (24 – 63)

Downregulation of FcεRIγ in CD56dim NK cells of cHBV patients

H D c H B V0

2 0

4 0

6 0

8 0

1 0 0

%F

cε

RI γ

+

****

%FcεRIγ+

adaptive NK cell

CD3ζ

NKG2C ↑

Helios ↓PLZF ↓

FcεRIγCD16

ADCC ↑↑

IFNγ ↓

Adaptive NK cells lack FcεRIγ expressionLee et al., Immunity 2015Schlums et al., Immunity 2015


p o s n e g p o s n e g 0

2 0

4 0

6 0

8 0

1 0 0

%F

cε

RI γ

+

*

*

HD cHBV

HCMV:

%FcεRIγ+


adaptive NK cell

CD3ζ

NKG2C ↑

Helios ↓PLZF ↓

FcεRIγCD16

ADCC ↑↑

IFNγ ↓


p o s n e g p o s n e g 0

2 0

4 0

6 0

8 0

1 0 0

%F

cε

RI γ

+

*

*

HD cHBV

HCMV: FcεRIγN

KG

2C

CD56dim NK cells of HCMV+ cHBV

conventionalNK cells

adaptive NK cells

%FcεRIγ+

adaptive NK cell

CD3ζ

NKG2C ↑

Helios ↓PLZF ↓

FcεRIγCD16

ADCC ↑↑

IFNγ ↓


FcεRIγ deficiency in CD56dim NK cells of CMV+ cHBV patients correlates with other protein downregulations

adaptive NK cell

CD3ζ

NKG2C ↑

Helios ↓PLZF ↓

FcεRIγCD16

Transcriptional regulation of adaptive FcεRIγ- NK cellsLee et al., Immunity 2015Schlums et al., Immunity 2015

Helios is down-regulated in FcεRIγ- NK cells in chronic HBV

FcεRIγ

Hel

ios


I

IIIII

IV

I II III IV0

2 0

4 0

6 0

8 0

1 0 0

% o

f C

D5

6d

im N

K c

ells

% FcεRIγ/Helios subsets

ADCC ↑↑

IFNγ ↓

FcεRIγ deficiency in CD56dim NK cells of CMV+ cHBV patients correlates with other protein downregulations

adaptive NK cell

CD3ζ

NKG2C ↑

Helios ↓PLZF ↓

FcεRIγCD16


Helios is down-regulated in FcεRIγ- NK cells in chronic HBV

FcεRIγ

Hel

ios


adaptive NK cells


I

IIIII

IV

% FcεRIγ/Helios subsets

I II III IV0

2 0

4 0

6 0

8 0

1 0 0

% o

f C

D5

6d

im N

K c

ells

ADCC ↑↑

IFNγ ↓

c o n v e n t io n a lN K c e lls

a d a p t iv eN K c e lls

0

2 0

4 0

6 0

8 0

1 0 0%

PL

ZF

+

****

%PLZF+

Are these FcεRIγ-Helios- CD56dim NK cells in CMV+cHBV patients adaptive NK cells?Analysis of the master regulator PLZF

PLZF

I

III



adaptive NK cells

adaptive NK cell

CD3ζ

NKG2C ↑

Helios ↓PLZF ↓

FcεRIγCD16

ADCC ↑↑

IFNγ ↓

Functional analysis (cytokine responsiveness)

IL-12/IL-15

IL-12/IL-18

c o n v e n tio n a lN K c e lls

a d a p tiv eN K c e lls

0

2 0

4 0

6 0

8 0

%IF

Ny+

****



0

2 0

4 0

6 0

8 0

% I

FN

y+

****

cytokine stimulation

%IFNγ+

%IFNγ+

adaptive NK cell

CD3ζ

NKG2C ↑

Helios ↓PLZF ↓

FcεRIγCD16

ADCC ↑↑

IFNγ ↓

Are these FcεRIγ-Helios- CD56dim NK cells in CMV+cHBV patients adaptive NK cells?



0

1 0

2 0

3 0

4 0

5 0

%IF

Ny+

**

CD16 crosslink

CMV-seropositive donors

adaptive NK cells ↑

CMV-induced adaptive NK cells are present in the majority of cHBV patients

HD: 80% 20%cHBV: 30% 70%

adaptive NK cell

conv.NK cell

conv.NK cell

adaptive NK cell

conv.NK cell

conv.NK cell

conv.NK cell

conv.NK cell

conv.NK cell

adaptive NK cell

conv.NK cell

adaptive NK cell

conv.NK cell

conv.NK cell

adaptive NK cell

conv.NK cell

conv.NK cell

conv.NK cell

conv.NK cell conv.

NK cell

conv.NK cell

conv.NK cell

conv.NK cell

conv.NK cell



cytokine responsiveness ↓IL-12/IL-15/IL-18 IL-12/IL-15/IL-18

IFNγ ↑↑

HD: 80% 20%cHBV: 30% 70%

adaptive NK cell

conv.NK cell

conv.NK cell

adaptive NK cell

conv.NK cell

conv.NK cell

conv.NK cell

conv.NK cell

conv.NK cell

adaptive NK cell

conv.NK cell

adaptive NK cell

conv.NK cell

conv.NK cell

adaptive NK cell

conv.NK cell

conv.NK cell

conv.NK cell

conv.NK cell conv.

NK cell

conv.NK cell

conv.NK cell

conv.NK cell

conv.NK cell

IFNγ ↓


IFNγ ↓



cytokine responsiveness ↓IL-12/IL-15/IL-18 IL-12/IL-15/IL-18

HD: 80% 20%cHBV: 30% 70%

adaptive NK cell

conv.NK cell

conv.NK cell

adaptive NK cell

conv.NK cell

conv.NK cell

conv.NK cell

conv.NK cell

conv.NK cell

adaptive NK cell

conv.NK cell

adaptive NK cell

conv.NK cell

conv.NK cell

adaptive NK cell

conv.NK cell

conv.NK cell

conv.NK cell

conv.NK cell conv.

NK cell

conv.NK cell

conv.NK cell

conv.NK cell

conv.NK cell

Functional dichotomy:

increased/conserved cytotoxicity decreased IFNγ productionIFNγ ↑↑


Novel insights into immune responses against HBV

CD8+ T cells NK cells

• memory-like• TCF1high

• promising target for PD1 blockade• determinant?

CD3ζ

NKG2C ↑

Helios ↓PLZF ↓

FcεRIγCD16

ADCC ↑↑

CMV


IFNγ

• expansion of epigeneticallyregulated adaptive NK cells

• driving force?• cross-talk with CD8+ T cells?

InhibitoryReceptors ↑

Proliferation ↓

CD39 ↓

CD127 ↑↑

Eomesdim

TCF1high

Robert ThimmeMaike Hofmann

Kathrin HeimCharlotte HerchenröderKatharina HerzogAlexander HohCatharina KramerTony MatschullaSebastian MerkerPatricia Otto-MoraAnita SchuchÖzlem SogukpinarCatrin TauberDominik WielandBritta ZecherXin Zhou

Christoph Neumann-Haefelin

Aurelien BeisertJanine KemmingMichael Kiraithe

Department of Medicine II - University of Freiburg

Acknowledgements

All patients and volunteers!

Tobias Böttler

Kristi BashoMaike SmitsKatharina Zoldan

Nico Büttner

Katharina GrützmannMiriam Krimmer

how does hbv interact with the immune...

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