PD-1, SOCS-1, Tim-3 in HCV infection
-WHY WE CARE?
Yao, Z. Q. M.D. Ph.D.
Director, Hepatitis (HCV/HIV) Program, JHQ-VAMC
Associate Professor, Division of Infectious Diseases
Department of Internal Medicine, Quillen COM ETSU
Disclosures
• Grant funding from NIH NIAID, NIDDK, and ETSU/WFU
• No other financial interests involved in this presentation
In this Presentation
We’ll talk:
1. Clinical features and immunodysregulations of HCV infection
2. Negative signaling molecules such as PD-1, SOCS-1, and Tim-3
in control of human innate to adaptive immune responses
We expect to know:
A) how HCV employ negative signaling molecules to establish
chronic infection;
B) why we care about this – its application in the HCV
pathogenesis, treatment, and vaccine development
Clinical features of HCV infection
15%
200 M WW
PD-1, SOCS-1, Tim-3
4 M U.S.
HIV
Why the majority of infected individuals become chronic?
Immunodysregulation in chronic HCV infection
Impaired Monocyte
maturation into DC
Decreased IL-12
Viral Persistence
T cell dysfunction
and exhaustion
mixed cryoglobulinemia
B cell clonal
expansion
non-Hodgkins lymphoma
B cell hyperactivation
Th17 cell and Foxp3+
Treg cell expansions
What is the underlying mechanism leading to these immunodysregulations?
Mechanism leading to these immunodysregulations
The primary site of HCV infection is within the liver, where hepatic sinusoids lack basal membrane with a very low velocity of blood flow
PD-1
PD-1
Tim-3
Tim-3+ CD14+ M/MØ
SOCS-1
100%
50%
0%
6h
12 h
HCV+ Huh-7
So HCV-infected hepatocytes has ample opportunity to contact circulating or infiltrating immune cells
24 h
48 h
HCV- Huh-7
What is PD-1
•
•
•
•
•
Programmed Death-1, first identified on apoptotic cells
Inducible expressed receptor on immune cells upon activation
Provides a negative signaling to TCR positive signaling pathway
A powerful negative feedback mechanism to balance the +/- signal
Blocking PD-1 signaling will reverse T cell dysfunction
PD-1 and T cell function / exhaustion
Tim-3 : a molecule different from PD-1
A new negative molecule first identified on Th1, but not Th2,
and now also found on other cell types: M/MФ, NK cells
Suppressor of cytokine signaling (SOCS)
– a family of negative inhibitors of cell signaling
Cytokine
Why we care about this?
-Negative signaling molecules in HCV pathogenesis
PD-1 & Tim-3 in Monocyte IL-12 regulation
Immunodysregulation in chronic HCV infection
Impaired Monocyte
maturation into DC
Decreased IL-12
Viral Persistence
Monocyte IL-12 expression is significantly suppressed in chronic HCV infection
Healthy
HCV
Gating strategy
CD14
15.2%
IL-12
50%
40%
30%
20%
10%
0%
60%
PD-1+ CD14+ cells
IL-12+ CD14+ cells
60%
**
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HCV-Infected HCV-Resolved
Healthy
50%
40%
30%
20%
10%
0%
**
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HCV-Infected HCV-Resolved
■
■
■
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■
■
■
■
■
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Healthy
Ma et al. Immunology 2010; Zhang et al. J Immunol 2011
PD-1 is inversely associated with IL-12 production by monocytes
B)
A)
Pearson Correlation = -0.464*
PD-1
PD-1+ CD14+ cells
LPS/R848
IL-12
50%
45%
40%
35%
30%
25%
20%
15%
10%
5%
0%
0%
C)
40%
IL-12 production
D)
50%
P = 0.003
CD14+ Cells
P = 0.034
40%
30%
20%
IL-12+
PD-1+ CD14+ Cells
50%
20%
10%
0
40%
30%
20%
10%
0
before
IFN/RBV
after
IFN/RBV
before
IFN/RBV
after
IFN/RBV
60%
Tim-3 is a negative molecule expressed on resting monocytes
to control IL-12 expression
un-stimulated
1.2%
45.8%
iso
TLR-stimulated
3.0%
Tim-3
Tim-3
iso
un-stimulated
2.7%
0.2%
55.3%
0.1%
0.4%
0.1%
TLR-stimulated
1.8%
2.2%
38.9%
IL-12
CD14
0.2%
0.3%
27.2%
Positive cells (%)
CD14
70
60
50
40
Tim-3
30
IL-12
20
10
IL-12
0
0h
6h
18h 24h 48h 72h
Time of TLR stimulation
Zhang et al. JLB 2011
Tim-3 functions as a break, and TLR as the driving force for IL-12 expression
Tim-3/IL-12 expression in resting and activated monocytes in HCV patients
A
Healthy Subject
Naïve
Activated
Isotype control
HCV Patient
Naïve
Activated
38.3
2.8
4.1
4.4
54.6
3.4
8.7
27.0
27.3
45.5
47.6
22.3
18.8
13.5
74.4
Tim-3
7.4
CD14
0.2
40.9
33.9
67.7
0.4
54.0
15.3
28.5
0.1
22.7
2.5
31.8
0.2
29.6
1.0
CD14
71.3
IL-12
**
B
***
C
NS
**
***
***
***
% of IL12+ CD14+ M/MØ
% of Tim-3+ CD14+ M/MØ
***
Naïve
Activated
Naïve
Activated
Healthy Subjects
Naïve
Activated
Chronic HCV Patients
Naïve
Activated
Zhang et al. PLoS One 2011
It’s not because of TLR expression, but due to defect of intracellular signaling
A)
Healthy Subjects
30
*
10
0
Healthy Subjects
HCV patients
99.5%
99.7%
40
20
Healthy Subjects
40.7%
TLR7+ CD14+ Cells
10.3%
TLR4+ CD14+ Cells
B)
HCV patients
100
50
0
Healthy Subjects
HCV patients
HCV patients
Healthy subject
HCV-infected
21.6%
D)
IgG
+
HCV-resolved
23.7%
9.6%
anti-Tim-3
+
Core
Phospho Stat1
Total Stat1
STAT-1+ CD14+ Cells
C)
30
20
*
10
0
Healthy HCV-infected HCV-resolved
HCV core
TLR
PD-1
Isotype
gC1qR
Count
LPS R848
Control IgG
+
11.7%
3.2%
LPS R848+core+a-PDL-1 8.9%
SOCS-1
IL-12
a-PDL-1
+
Core
SOCS-1
β-actin
% IL-12+CD14+ cells
M/MФ IL-12
production
LPS R848+core+IgG
1.2%
14
12
**
10
8
**
6
4
2
0
LPS/R848
L/R core contl IgG L/R core anti-PDL-1
Silencing SOCS-1 inhibits PD-1 expression and improve IL-12 production
A)
48 h after transfection
Control siRNA
SOCS-1 siRNA
+
+
72 h after transfection
Control siRNA
+
SOCS-1 siRNA
+
Core
SOCS-1
β-Actin
B)
Count
Isotype 0.73%
LPS R848 Core control siRNA 39.1%
LPS R848 Core SOCS-1 siRNA 2.9%
PD-1
C)
Count
Isotype 2.88%
LPS R848 Core control siRNA 10.1%
LPS R848 Core SOCS-1 siRNA 19.4%
IL-12
Crosstalk between PD-1 and SOCS-1 to inhibit STAT-1/5 phosphorylations
Control siRNA
+
+
Control IgG
-
+
+
a-PDL-1
+
pSTAT-1
Total STAT-1
Total STAT-1
Control IgG
Anti-PDL-1
Anti-PDL-1
%STAT-1+CD14+ cells
CD14
25
*
20
15
10
5
0
L/R core contl IgG
Core
pSTAT-1
STAT-5
Control IgG
Core
STAT-1
D)
B)
SOCS-1 siRNA
L/R core anti-PDL-1
CD14
%STAT-5+CD14+ cells
A)
**
80
70
60
50
40
30
20
10
0
L/R core contl IgG
L/R core anti-PDL-1
Our Model
LPS/R848
TLRs
Signaling pathways for
IL-12 expression (Jak/STAT)
Gal-9
Tim-3
PD-L1
HCV core
PD-1
gC1qR
SOCS-1
Th1/Tc1 dysregulation
Viral persistence
Viral clearance
Immunodysregulation in chronic HCV infection
Impaired Monocyte
maturation into DC
Decreased IL-12
Viral Persistence
T cell dysfunction
/ exhaustion
B cell clonal
expansion
mixed cryoglobulinemia
non-Hodgkins lymphoma
B cell hyperactivation
What is the underlying mechanism leading to these immunodysregulations?
Why we care about this?
-Negative signaling molecules in HCV pathogenesis
HCV infection lead to a differential effect on T/B lymphocytes what is the underlying mechanism ?
45.3%
HCV-NHL
10.4%
19.1%
96.2%
IgM
5.8%
IgG
HCV-Tetramer
CD69
19.4%
TALL-1
Cell Immunology & Biology 2011
83.3%
HCV-NHL
4.2%
72.3%
48.8%
HS
CD4
CD8
CD4
CD20
70.0%
53.5%
Tetramer CD8
HCV
CD20
6.9%
PD-1
HCV-NHL
PD-1
PD-1
47.3%
37.2%
HS
HCV-NHL
13.7%
HS
CD20
CD20
Differential regulation of T/B lymphocyte activation in patients with HCV-NHL
T cells
____________
B cells
____________
HCV-NHL HS
HCV-NHL HS
T cells
____________
B cells
____________
HCV-NHL HS
HCV-NHL HS
SOCS-1
SOCS-1
hβ2M
pSTAT1
Differential regulation of T / B lymphocyte signaling by HCV core protein
T cells
+
-
B cells
+
-
Core
SOCS-1
-Actin
T cells
+
-
B cells
+
-
Core
SOCS-1
-Actin
Conclusion: HCV induces a differential regulation of PD-1/SOCS-1 expression, which translate into
a differential regulation of T/B lymphocyte functions through Jak/STAT pathway
Blocking PD-1 signaling restores T cell activation and proliferation
Anti-PD-L1
Control Ab
29.8%
17.1%
B)
T cell Counts
CD69
A)
Anti-PD-L1
80% 10% 9%
Control Ab
1%
46% 13% 36% 5%
HS
HCV-Tetramer
CD4
21.3%
13.7%
CD8
22% 36% 38% 4%
1%
9% 77%
CFSE
13%
HCV-NHL
Why we care about this?
-Negative signaling molecules in HCV pathogenesis
Differential regulation of IL-12/IL-23 expressions by
M/MФ leads to TH17 cell and Foxp3+ Treg development
Differential regulation of IL-12/IL-23 expressions by M/MФ
leads to TH17 cell development during HCV infection
**
HS
*
IL-12 p35
HCV
IL-23 p19
HS
**
Pearson r=0.465
p＜0.05
IL-17A
HCV
CD4
IL-23/IL-12 by CD14+ cells
Hepatocyte
Hepatocyte
Hepatocyte
TLR
TLR
monocyte
 Tim-3
STAT-3
STAT-1
 IL-23
HCV
 IL-12
Tim-3
 Foxp3+ Tregs
 TH17
Gal-9
Differential regulation of IL-12/IL-23 expressions by M/MФ leads to
TH17 cell and Foxp3+ Treg development during HCV infection
HS
A)
HCV
**
8.91
11.94
13.54
20.85
74.75
4.40
53.68
11.93
B)
HS
HCV
***
1.54
5.68
2.44
9.41
52.82
39.95
62.70
25.45
*
A)
B)
HS
*
HCV
HS
HCV
25.86
15.78
20.06
7.42
15.94
4.89
14.53
7.26
28.50
29.85
45.08
27.44
62.47
16.7
59.18
19.03
NS
**
NS
Pearson Correlation = 0.75
Sig. (1-tailed)=0.0002; (2-tailed)=0.0004
Foxp3+ cells in CD4+CD25+ cells
14
12
10
8
6
4
2
0
0
CD4+
CD4+
CD25+
CD4+
CD25+
Foxp3+
CD4+
CD25+
Foxp3-
10
20
30
40
50
Tim-3+CD4+CD25+Foxp3+ cells
60
Differential regulation of IL-12/IL-23 expressions by monocytes/macrophages
leads to TH17 cell and CD4+CD25+Foxp3+development during HCV infection
HCV-infected hepatocytes
produce Gal-9 and TGF-β
 IL-23/IL-12
 IL-17
CD4+
T cell
CD25+
FoxP3T eff
 TGF-β/IL-10
CD25+
FoxP3+
T reg
Tim-3 is up-regulated more
on Foxp3+ Tregs than on Teffs
 IL-2
 proliferation
 apoptosis
 apoptosis
 proliferation
Tim-3/Gal-9 interactions shift the
balance of Tregs/Teffs by regulating
T cell proliferation and apoptosis
Teff
Treg
Treg
α-Tim-3
Moorman JP et al J Immunol 2012
Tim-3
/Gal-9
Treg
α-Tim-3 may correct the imbalance
of Tregs/Teffs ratio induced by HCV
Immunodysregulation during chronic HCV infection
HCV-infected
Hepatocytes
Impaired CD14+ M/MΦ
maturation into DC
Accumulated TH17 &
Foxp3+ Treg cells
Increased PD-1,
SOCS-1, Tim-3
Decreased IL-12
Increased IL-23
Increased IL-17
HCV chronic infection
Increased IL-10
Increased TGF-β
Autoimmune disorders
Diminished CD4+/ CD8+
T cells
Decreased IL-2
Decreased TNF-α
Decreased IFN-γ
Aberrant CD19+ B cell
activation
Increased IgG
Increased IgM
Why we care about this?
-Negative signaling molecules in Vaccine response
HBV vaccine response and HCV vaccine development
Tim-3 on HBV vaccine failure in HCV-infected individuals
HBV Vaccine response: 90% in Healthy Subjects; 50% in HCV-infected patients
A)
***
HBV-R
**
Isotype
% Tim-3/CD14+ cells
HBV-NR
% IL-23p19/CD14+ cells
% IL-12p35/CD14+ cells
B)
*
PD-1/SOCS-1 on HBV vaccine failure in HCV-infected individuals
12.1% vs 7.0%, P=0.002
18.00%
5.6% vs 4.5%, P>0.05
16.00%
14.00%
CD69+ CD4+ T cells
12.00%
10.00%
8.00%
6.00%
4.00%
2.00%
100.00%
90.00%
80.00%
70.00%
60.00%
50.00%
40.00%
30.00%
20.00%
10.00%
0.00%
0.00%
Pearson Corr. = - 0.374**
Sig.(2-tailed) = 0.001
10.00%
20.00%
PD-1+
0.00%
CD4+
30.00%
T cells
HCV patients HCV patients HCV resolved
Healthy
HBV-NR (n=29) HBV-R (n=32) individuals(n=6) Subjects(n=10)
HBsAg stimulation
HBV-R
HBV-NR
a-CD3/28 stimulation
HBV-R
0.4
HBV-NR
SOCS-1
β-actin
SOCS-1/actin
PD-1 expression on CD4+ T cells
9.4% vs 4.9%, P=0.007
*
0.3
*
0.2
0.1
0
HBV-R
HBV-NR
HBsAg stimulation
HBV-R
HBV-NR
a-CD3/28 stimulation
40.00%
Control Ab
80
18.1%
29.9%
HBsAg stimulation
31.6%
57.8%
% CD69+ in CD4+ T cells
A)
a-PD-L1 Ab
a-CD3/28 stimulation
70
60
IgG
*
a-PDL-1
50
*
40
30
20
10
0
Control Ab
0% 0.3% 7% 92%
a-PD-L1 Ab
1.5% 8% 18% 73%
HBsAg stimulation
100
80
60
IgG
a-PDL-1
40
*
20
*
0
M1
100
0% 1.5% 19% 79%
1%
21% 58% 20%
a-CD3/28 stimulation
CFSE / a-CD3/28
B)
CFSE / HBsAg
HBsAg stimulation a-CD3/28 stimulation
80
60
M2
M3
M4
IgG
*
a-PDL-1
40
*
20
*
0
M1
M2
M3
M4
Why do we care - Listeria monocytogenes (Lm)-based DC-targeting HCV vaccine Development
HCV vaccine development: HCV-quasispecies; HCV-delivery; HCV-models; HCV-exhaustion
Listeria
monocytogenes
Lm vector
HCV
antigens
Virulence
determinants
NS5B
∆actA/∆inlB
Lm-infected DC
Lm-NS5B
CD14
Monocyte
iDC induced by
GM-CSF + IL-4
mDC induced by
TNF-α + Poly I:C
mDC infected by
Lm-HCV vaccine
HLA-ABC
HLA-DR
CD209
CD1a
CD80
CD83
CD86
%Tim-3+ cells
%IL-12 + cells
Why do we care ?
Improve Lm-based DC-targeting HCV vaccine by blocking Tim-3 signaling
M/MФ
BSA-FITC Uptake (ΔMFI)
iDC
mDC
Un-infected
HCV HCV+IgG HCV+a-Tim-3
Iso
Gating strategy
HCV-resolved
CD3+CD8+
Tim-3
Lm-control
IgG
α-Tim-3
Lm-NS5B
IgG
α-Tim-3
CD3+CD8+
%HCV-Tet+/CD3+CD8+
HCV-Tetramer
B)
HCV-infected
SSC
HCV-Tetramer
A)
%Tim-3+/HCV-Tet+CD3+CD8+
Why do we care?
Improve Lm-based DC-targeting HCV vaccine by blocking Tim-3 signaling
Why do we care?
Listeria monocytogenes (Lm)-based DC-targeting HCV vaccine
B)
Granzyme-B
%GranzymeB+/HCV-Tet+CD3+CD8+
IFN-γ
%IFN-r+/HCV-Tet+CD3+CD8+
A)
Why do we care?- novel therapeutics
MHC/peptide/B7
LPS
TLR
CD3
CD28
Gal-9
PD-L1
HCV Core
Tim-3
PD-1
gC1qR
STAT
Monocyte IL-12
T cell activation
α-gC1qR
α-PD-1
SOCS
α-HCV core
α-Tim-3
Negative T cell regulators
T cell dysfunction
Improve HBV vaccine response
in HCV/HIV-infected individuals
viral clearance
HCV persistence
Improve HCV - DC
therapeutic Vaccine
Acknowledgements
Dr. T. Niki: President of GalPharm, Japan;
Dr. T.J. Liang, Chief Liver Dis, NIH NIDDK
Dr. T Wakita, Director Virology Lab, NIH, Japan;
Dr. D. Brockstedt, VP of Aduro BioTech, CA