Is it possible to restore innate and adaptive

Gabriele Missale

Unit of Infectious Diseases and HepatologyLaboratory of Viral Immunopathology

Azienda Ospedaliero-Universitaria di Parma, Italy

HBV and T cell Exhaustion

INTRAHEPATIC INHIBITORY MECHANISMS

Modified from U. Protzer et al. Nature Reviews in Immunology 2012

List of topics

- HBV-specific T cells in chronic infection

- NK cell response and its regulatory role on HBV-specific T-cell response

- Potential strategies to reconstitute the anti-viral T cell function

- Molecular basis of CD8 cell dysfunction in chronic HBV infection

HBeAg(+) HBeAg(-) / anti-HBe(+)

ALT

HBV DNA

Absent/Minimal CH

Moderate to severe CH

Moderate to severe CHRemission

Cirrhosis

Immunotolerantphase

Immuno-activephase

Inactive phaseLow replication

Reactivation phase

Cirrhosis

109-1012 IU/mL >2000-<109 IU/mL <2000 IU/mL >2000 IU/mL

Inactive cirrhosis

Adapted from Fattovich G. Sem Liver Dis. 2003

HBsAg

Occult infection

The five phases of the natural history of chronic HBV infection

*<200 IU/mL

HBV-specific T cells in chronic infection

Different levels offunctional Tcell efficiency indifferentconditions ofHBVcontrol

Boni C, Laccabue D et al. Gastroenterology 2012;143:963–973

IFN-γ IL-2 TNF-α30

25

20

15

10

5

030

25

20

15

10

5

0

Mean%IFN-γ,IL-2an

dTN

F-α

+/Tcells

CD4+Tcellresponses

CD8+Tcellresponses

Acutehepatitis B

(resolution phase)

Inactivecarriers

Occultinfections

NaïveHBeAgnegativeCHB

HBV-SPECIFIC CD8 CELLS ARE PREFERENTIALLY CONCENTRATED WITHIN THE LIVER IN PATIENTS WITH

CHRONIC HBV INFECTION(Fisicaro P. et al. Gastroenterology 2010)

%H

BV-

TET+

/CD

8+

00.020.040.060.080.1

1246810 LIVER BLOOD

HBV-DNA TITER IU/ml

Mea

n%

TET+

/CD

8+

intr

ahep

atic

cells

0123456

0-104 104-105 >105

p=0.006

p=0.03

p=0.0002

0.5

1.5

0

1.0

IFN-γ IFN-γ/IL2

CD4

AND

CD8

T CE

LLS

00.20.40.60.81.01.21.41.6

1x100 1x102 1x104 1x106 1x108 1x1010

HBV LIVER

HBV-DNA IU/ml

%IF

N-γ

CD

4+C

D8

cont

r

r = - 0.72p = 0.014

p=0.0078BLOOD

LIVER

INTRAHEPATIC HBV-SPECIFIC T CELLS ARE MORE DEEPLY EXHAUSTED THAN THEIR PERIPHERAL BLOOD COUNTERPARTS IN CHRONIC HBV INFECTION

(Fisicaro P. et al. Gastroenterology 2012)

TNF-α

p=0.01p=0.02

Is it possible to restore innate and adaptive immunity for HBV?

PUTATIVE MECHANISMS OF T CELL EXHAUSTION IN HBV INFECTION

High viral load with massive production of secretory proteins (HBsAg, HBeAg)

Tolerizing liver environmentSinusoid

Space of Disse

Hepatocyte

Naïve CD8 cell

Effector CD8 cell

Chronic HBV infection

Inefficient T cell function/differentiation

+Ag

Efficient T cell function/differentiationAcute Self-limited Infection

Effector memory

Central memory

Rapid Proliferation/ Differentiation

T CELL FUNCTIONAL IMPAIRMENT IN CHRONIC HBV INFECTION

Naïve CD8 cell

Effector CD8 cell

+Ag

Efficient T cell function/differentiationAcute Self-limited Infection

Effector memory

Central memory

Rapid Proliferation/ Differentiation

CAN THE HBV-SPECIFIC T CELL FUNCTION BE RESTORED BY ANTIGEN DECLINE IN CHRONIC HBV PATIENTS?

Ant

igen

de

clin

e?

Restored T cell function/differentiation

Effect of long-term NUC therapy on T cell responses

0

2

4

6

8

10

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

NUC treated patients with persistent HBV-DNA suppression but HBsAg positive

MIXx core env pol

Mea

n%

IFN

γ+/T

cel

ls

NUC treated patients with complete control of infection (HBV-DNA and HBsAg negative)

0

2

4

6

8

10

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

MIXx core env pol

Mea

n%

IFNγ+

/T c

ells

IFNγ/CD8+ IFNγ/CD4+

Stable restoration of the T cell function after long-lastingsuppression of HBV replication induced by NUC therapy and

detected following expansion in vitro

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 160

2

4

6

8

10

x core env pol

Anti-HBe+ chronic infection

Mea

n %

IFN

γ+/T

cel

ls

0

2

4

6

8

10

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16x core env pol

Resolution phase of acute HBV infection

Mea

n%

IFNγ+

/T c

ells

Boni C. et al. Gastroenterology 2012

A stable restoration of the T cell function is detectable upon in vitro expansion in NUC treated patients after long-lasting

suppression of HBV replication

100

0

20

40

60

80

p=0.0001

p<0.0001

CMV

HBV

FLU

% P

D1+

mul

timer

cells

Dext FLU

43

2.4%

42 3

PD1

Dext HBV43

97.5%

43

Chronic HBV treated patient

Tcellspecificity

PD1

Chronic HBVtreated patients

PD-1 expression by HBV-specific CD8 cells in NUC treated patients

Boni C. et al. Gastroenterology 2012

CD107a

NUC treatedHBsAg neg

NUC treatedHBsAg pos

Acute hepatitis B(follow-up)

IL-2

IFN-γ

% o

f HBV

dex

tram

er+

CD

8 T

cells

prod

ucin

g

020406080

100

012345

60

0

20

40

60

80

100

In vitro

medium peptide

NUC treated

HBsAg neg

Acute hepatitis B

(follow-up)

IFN-γ

0% 0%

0% 29%

Dext core

CMV

FLU

IFN-γ

CD

8 Dext FLU

Dext CMV

1% 72%

0% 88.7%

CD

8

medium peptide

Dext core

T cell restoration following NUC treatment is efficient in vitro Boni C. et al. Gastroenterology 2012

Naive CHB

CD107a

NUC treatedHBsAg neg

NUC treatedHBsAg pos

Acute hepatitis B(follow-up)

IL-2

IFN-γ

% o

f HBV

dex

tram

er+

CD

8 T

cells

prod

ucin

g

020406080

100

012345

60

0

20

40

60

80

100

In vitro

medium peptide

NUC treated

HBsAg neg

Acute hepatitis B

(follow-up)

IFN-γ

0% 0%

0% 29%

Dext core

CMV

FLU

IFN-γ

CD

8 Dext FLU

Dext CMV

1% 72%

0% 88.7%

CD

8

medium peptide

Dext core

T cell restoration following NUC treatment is partial ex vivo Boni C. et al. Gastroenterology 2012

Naive CHB

Ex vivo

0

100

200

300

400

500

600

700

800

900

NUC treatedHBsAg neg

6 pts

NUC treatedHBsAg pos

5 pts

Acute hepatitis B(follow-up)

5 pts

Naive HBeAgnegative CHB

15 pts

05

10152025303540

% positive responses

Mea

n D

elta

spot

s/ 20

0,00

0 T

cells

Ex vivo frequencies of IFN-γ producing HBV-specific T cells after peptide stimulation

(ELISPOT analysis)

p=0.007

p=0.004

p=0.0004

Boni C. et al. Gastroenterology 2012

NK cell function in NUC treated patients

Is functional HBV-specific T cell restoration associatedwith a parallel improvement of NK cell function in NUC

treated patients?

Functional NK cell dichotomy in chronic HBV infection Impaired IFN-γ production with normal cytotoxicity

Peppa D. et al Plos Pathogens 2010

IFN-γ

CD107a

N=29Healthy

N=46CHB%

IFNγ

prod

uctio

n N

K T

otal

P<0.000125

20

15

10

5

0

% C

D10

7 pr

oduc

tion

NK

Tot

al

N=33CHB

N=21Healthy

0

20

40

60

80

100 0.0233

%IF

Ng+

/CD

56 b

right

0

20

40

60

%C

D10

7a/N

Kdi

m

Boni C. et al. Hepatology 2015; 62:1697-709

59%0%

CD56

+CD3

-

IFN-γ

medium IL12 + IL18

IFN-γ production by NK cells is not significantlyimproved by NUC therapy

0

20

40

60

80

100ns

%IF

Nγ+

/CD

56 b

right

Boni C. et al. Hepatology 2015; 62:1697-709

CD56

+CD3

- 43%1.8%11%1%

CD107a

E+T+IL15E+IL15E+TEffectors ALONE

Capacity to degranulate of NK cells is notsignificantly affected by NUC treatment

0

20

40

60ns

%C

D10

7a+/

CD

56

dim

Boni C. et al. Hepatology 2015; 62:1697-709

% global NK cell function

%TNF-α/CD8+

%TNF-α/CD4+

%IFN-γ/CD8+

%IFN-γ/CD4+

%IL-2/CD8+

%IL-2/CD4+

% global T cell function

%IFN-γ%CD107a

0

50

100

50

100

150

150

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16Patients

CD107and

Reciprocalbehaviour ofNKcellandHBV-specificTcellresponsesInNUC-treatedpatients

Boni C. et al. Hepatology 2015; 62:1697-709

% global NK cell function

%TNF-α/CD8+

%TNF-α/CD4+

%IFN-γ/CD8+

%IFN-γ/CD4+

%IL-2/CD8+

%IL-2/CD4+

% global T cell function

%IFN-γ%CD107a

0

50

100

50

100

150

150

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16Patients

Reciprocalbehaviour ofNKcellandHBV-specificTcellresponsesInNUC-treatedpatients

Boni C. et al. Hepatology 2015; 62:1697-709

global HBV-specific T cell function

0 50 100 1500

50

100

150

0.0128p=

-0.6357r=

glob

al N

K ce

ll fu

nctio

n

InversecorrelationbetweenglobalNKcellandglobalHBV-specificTcellfunctionsin16NUC-treatedpatients

Boni C. et al. Hepatology 2015; 62:1697-709

0 5 10 15 20 250

20

40

60

80

0 10 20 30 400

20

40

60

80

0 5 10 150

20

40

60

80

r -0.7251p = 0.001

0 10 20 30 40 500

20

40

60

80

0 5 10 15 20 250

20

40

60

80

0 5 10 150

20

40

60

80

r -0.5505p = 0.035

r -0.6941p = 0.0037

r -0.5149p = 0.039

ns

ns

%IF

N-γ+

/CD5

6 br

ight

%IF

N-γ+

/CD5

6 br

ight

%IF

N-γ+

/CD5

6 br

ight

%IF

N-γ+

/CD5

6 br

ight

%IF

N-γ+

/CD5

6 br

ight

%IF

N-γ+

/CD5

6 br

ight

%IFN-γ/CD4+HBV-specific T cells

%TNF-α/CD4+HBV-specific T cells

%IL-2/CD4+HBV-specific T cells

%IFN-γ/CD8+HBV-specific T cells

%TNF-α/CD8+HBV-specific T cells

%IL-2/CD8+HBV-specific T cells

InversecorrelationbetweenNKcellIFN-γ productionandHBV-specificTcellcytokineproductioninNUC-treatedpatients

0

0,5

1

1,5

2

2,5

3

1 2 3 4 5 6 7 8 9 10

PBMC NKdepleted

+NK0.0

0.5

1.0

1.5 0.003 0.03

0

0,5

1

1,5

2

2,5

3

1 2 3 4 5 6 7 8 9 10

PBMC

NK DEPLETED

+ NK (day 0)

HBV-Core and Polymerase

% G

loba

l HBV

-spe

cific

CD

4 cy

toki

ne p

rodu

ctio

n

% G

loba

l HBV

-spe

cific

CD

8 cy

toki

ne p

rodu

ctio

n

PBMC NK DEPLETED

+NK (day 0)

0.0

0.5

1.0

1.50.003 0.03

% C

D4+

and

CD8+

cyto

kine

+ T

cells

ImprovementofHBV-specificTcellfunctionafterNKcelldepletion bycellsortinginNUC-treatedpatients(1)

NUC-treated patients

0% 0%HBV-POL

295-315

IFN-γ

0.3%

PBMC NKDEPLETED (Δ NK) +NK(DAY0)

HBV-POL 150-191

TNF-α

0.8% 0%0.07%

CD4

CD8

0.00.10.20.30.40.5

PBMC NKdepleted

+NK

% G

loba

l HBV

-spe

cific

CD

8 cy

toki

ne p

rodu

ctio

n

0

5

10

15

20

25

30

1 2 3 4 5 6

PBMCNK DEPLETED+ NK (day 0)

CMV, EBV, FLU

0

5

10

15

20

25

30

1 2 3 4 5 6

PBMCNK DEPLETED+ NK (day 0)

% G

loba

l HBV

-spe

cific

CD

4 cy

toki

ne p

rodu

ctio

n

% C

D4+

and

CD8+

cyto

kine

+ T

cells

PBMC NK DEPLETED

n.s.

0

10

20

30

NUC-treated patientsNUC-treated patients

NKcelldepletiondoesnotaffectthefunctionofTcellsofdifferentspecificity inNUC-treatedpatients

CD4 CD8

PBMC NKdepleted

012345

PBMC NKdepleted

05

101520

n.s.n.s.

Boni C. et al. submitted

List of topics

- HBV-specific T cells in chronic infection

- NK cell response and its regulatory role on HBV-specific T-cell response

- Potential strategies to reconstitute the anti-viral T cell function

- Molecular basis of CD8 cell dysfunction in chronic HBV infection

Expression of various inhibitory receptors on circulating and intrahepatic virus-specific CD8 cells of patients with

chronic HBV infectionAPC

T CELL

B7 FAMILY TNF SUPERFAMILY

B7.1 or B7.2

PD-L1PD-L2

PD-1 CTLA-4 CD28

ICOSL

ICOS

INHIBITION

OX40L 4-1BBLCD40CD70

OX40 CD27CD40L 4-1BB

COSTIMULATORY SIGNALS

0

10

20

30

40

50

60

70

80

90

100

LAG-32B4PD-1 CD137CD160 CTLA-4

p = 0.0003

p = 0.011

p = 0.0156

%C

OST

IM.+

/TET

+

Liver Blood

Fisicaro P. et al. Gastroenterology 2012

Expression of PD-1 and CD127 on circulating and intrahepatic virus-specific CD8 cells of patients with chronic HBV infection and

effect of PD-1/PD-L1 blockade on the T cell function

Fisicaro P. et al. Gastroenterology 2010, 2012

%PD

-1+/

HB

V-TE

T+LIVER BLOOD

0

20

40

60

80

100

p=0.0039

%C

D12

7/H

BV-

TET+

LIVER BLOOD

0

20

40

60

80

100

p=0.0039

BLOOD

0

0.5

1

2.5

3

CONTROL Anti-PD-L1

p=0.0290

0.5

1

2.5

3

p=0.012

%IF

N-γ

+/C

D8+

CD

3+

LIVER

CONTROL Anti-PD-L10

0.5

1

2.5

3

p=0.02

0

0.5

1

2.5

3

p=0.04

%IF

N-γ

+/C

D4+

CD

3+

05

101520253035404550

p=0.01

fold

incr

ease

LIVER BLOOD

Strategies based on the correction of individual dysregulatedpathways can only induce a partial restoration of the T cell function

MESSAGE

HBV-specific T cells

Genome-wide expression profiling

Mysregulated genes and pathways associated with T

cell exhaustion

Correction strategies to restore anti-viral T cell

functions

TRANSCRIPTOME STUDY IN ACUTE, RESOLVED AND CHRONIC HBV INFECTION

Isolation of HBV/FLU-specific

CD8+ T cellsby cell sorting

RNA extraction and amplification

Gene expressionby microarray analysis

(4x44K Agilent)

VALIDATION AND DISCOVERY OF NEW TARGETS

Patient infection ALTA1 ACUTE 1785

A2 ACUTE 998

A3 ACUTE 659

A4 ACUTE 1118

A5 ACUTE 211

R1 RESOLVED HEPB 16

R2 RESOLVED HEPB 17

R3 RESOLVED HEPB 20

R4 RESOLVED HEPB 12

CH1 CHRONIC 40

CH2 CHRONIC 96

CH3 CHRONIC 68

CH4 CHRONIC 63

CONTROLS CELLSPECIFICITY

H1 HEALTHY FLU

H2 HEALTHY FLU

H3 HEALTHY FLU

H4 HEALTHY FLU

H5 HEALTHY FLU

Pre-sorting Post-sortingAcute

Resolved

HBV Dextramer

CD

8

Chronic

97.5%0.93%

0.10% 98.7%

0.04% 98.2%

Visualization of overall sample distribution and distances between groups of patientsby principal component analysis (PCA)

Acute

Chronic

Resolved

TRANSCRIPTOME STUDY IN ACUTE, RESOLVED AND CHRONIC HBV INFECTION

Fisicaro P. et al. submitted

Results and conclusion of molecular analysis of HBV-specific CD8 T-cells

1. Downregulation of genes involved in mithocondrion function2. Dna repair3. Proteasome

Exhausted HBV-specific CD8 cells are deeply impaired at a metabolic and energetic level with a prevalent down-regulation of various core cellular processes centered on extensivemitochondrial alterations.

A significant improvement of mitochondrial and antiviral CD8 functions was elicited by mitochondrion-targeted antioxidants

Main message

A deep metabolic and energetic impairment istypical of exhausted T cells

Evidence

Multiple levels of correction will likely be needed to restore an efficient anti-viral T cell function

(unless correction affects specific core central processes ableto indirectly affect other distal regulatory pathways)

Question

Is restoration of an efficient anti-viral T cellfunction an achievable objective?

AcknowledgmentsC. BoniV. Barili

D. LaccabueL. Talamona

M. RossiC. Cavallo

Laboratory Viral ImmunopathologyUnit Infectious Diseases and Hepatology

Azienda Ospedaliero-Universitariadi Parma, Italy

P. FisicaroA. PennaM. Pilli

A. OrlandiniT. Giuberti

C. Mori

G. Missale C. Ferrari

P. Lampertico M. Levrero

Cancer Research Center of LyonDivision of GastroenterologyUniversity of Milan, Italy