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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