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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
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
We’ll talk:
200 M WW
4 M U.S.
Clinical features of HCV infection
15%
Why the majority of infected individuals become chronic?
PD-1, SOCS-1, Tim-3
HIV
What is the underlying mechanism leading to these immunodysregulations?
T cell dysfunction
and exhaustion
mixed cryoglobulinemia
non-Hodgkins lymphoma
B cell clonal expansion
Viral Persistence
Immunodysregulation in chronic HCV infection
B cell hyperactivation
Decreased IL-12 Impaired Monocyte maturation into DC
Th17 cell and Foxp3+ Treg cell expansions
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
So HCV-infected hepatocytes has ample opportunity to contact circulating or infiltrating immune cells
PD-1
SOCS-1
Tim-3
PD-1
6 h 12 h 24 h 48 h Ti
m-3
+ CD
14+ M
/MØ
HCV+ Huh-7 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-1A 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
Viral Persistence
Immunodysregulation in chronic HCV infection
Decreased IL-12 Impaired Monocyte maturation into DC
Gating strategy
Healthy HCV
0%
10%
20%
30%
40%
50%
60%
▲
▲
▲▲
▲
▲▲▲
□
□
□
□
□
□
□
□
■
■■
■
■
■■■
■■■
■
□
____________________________ **
IL-1
2+ C
D14
+ c
ells
▲▲
▲
▲▲▲▲▲▲▲▲▲
_______________*
□
HCV-Infected HCV-Resolved Healthy
▲
▲
▲▲
▲
□□
□□□
■
■
Ma et al. Immunology 2010; Zhang et al. J Immunol 2011
Monocyte IL-12 expression is significantly suppressed in chronic HCV infection
IL-12
CD
14
□■
▲
0%
10%
20%
30%
40%
50%
60%
□
__________________________ **
PD
-1+
CD
14+ c
ells
▲▲
▲
▲▲▲▲
▲▲
▲
▲
▲
▲▲▲▲
▲▲
▲■
■■
■■■■■■
■■
□□
□
□□
□□
□
____________ *
HCV-Infected HCV-Resolved Healthy
▲ ▲▲▲▲
■■□□□□
□
15.2%
PD
-1+ C
D14
+ c
ells
Pearson Correlation = -0.464*
PD
-1+ C
D14
+ C
ells
10%
0
20%
30%
40%
50%
10%
0
20%
30%
40%
50%
IL-1
2+ C
D14
+ C
ells
before after IFN/RBV IFN/RBV
before after IFN/RBV IFN/RBV
B)
C) D)
P = 0.003 P = 0.034
PD-1 is inversely associated with IL-12 production by monocytes
IL-12
PD
-1
IL-12 production
LPS/R848
A)
iso un-stimulated TLR-stimulated
0.3% 27.2%
45.8% 3.0%
CD14
Tim
-3
IL-12
CD14
0.2%
1.2%
Tim-3 is a negative molecule expressed on resting monocytes to control IL-12 expression
Tim-3 functions as a break, and TLR as the driving force for IL-12 expression
Tim
-3
55.3% 0.4%
0.1%
1.8% 2.2%
38.9%
IL-12
2.7% 0.2%
0.1%
Zhang et al. JLB 2011
Time of TLR stimulation
Pos
itiv
e ce
lls (
%)
iso un-stimulated TLR-stimulated
Naïve Activated Naïve Activated
*** ***
**
**
% o
f Tim
-3+ CD
14+ M
/MØ
Naïve Activated Naïve Activated
Healthy Subjects Chronic HCV Patients
*** ***
***
NS
% o
f IL1
2+ CD
14+ M
/MØ
B C
A
7.4 38.3
27.0 27.3
2.8 4.1
45.5 47.6
4.4 54.6
22.3 18.8
3.4 8.7
13.5 74.4
71.3 0.2
28.5 0.1
40.9 33.9
22.7 2.5
67.7 0.4
31.8 0.2
54.0 15.3
29.6 1.0
IL-12
Tim
-3CD14
CD14
Isotype control Naïve Activated Naïve ActivatedHealthy Subject HCV Patient
Tim-3/IL-12 expression in resting and activated monocytes in HCV patients
Zhang et al. PLoS One 2011
It’s not because of TLR expression, but due to defect of intracellular signaling
TLR4
+ CD
14+ Ce
lls
Healthy Subjects HCV patients
10.3% 40.7%
*
Healthy Subjects HCV patients
Healthy Subjects HCV patients
99.7% 99.5%
TLR7
+ CD
14+ Ce
lls
Healthy Subjects HCV patients A) B)
STAT
-1+ C
D14
+ Ce
llsHealthy subject HCV-infected HCV-resolved
21.6% 9.6% 23.7%
Healthy HCV-infected HCV-resolved
C)
*
D)
Phospho Stat1
Total Stat1
+ + Core
IgG anti-Tim-3
gC1qR PD-1
TLR
M/MФ IL-12 production
HCV core
SOCS-1
+ + Core Control IgG a-PDL-1
SOCS-1
β-actin
IL-12
Cou
nt
Isotype 1.2%
LPS R848 11.7%
LPS R848+core+IgG 3.2%
LPS R848+core+a-PDL-1 8.9%
* *
% I
L-1
2+C
D14
+ c
ells
* *
48 h after transfection 72 h after transfection
Control siRNA SOCS-1 siRNA Control siRNA SOCS-1 siRNA
SOCS-1
β-Actin
+ + + + Core
A)
B)
C)
Isotype 0.73%
LPS R848 Core control siRNA 39.1%
LPS R848 Core SOCS-1 siRNA 2.9%
Isotype 2.88%
LPS R848 Core control siRNA 10.1%
LPS R848 Core SOCS-1 siRNA 19.4%
PD-1
Cou
nt
IL-12
Cou
nt
Silencing SOCS-1 inhibits PD-1 expression and improve IL-12 production
+ + Core Control siRNA SOCS-1 siRNA
pSTAT-1
Total STAT-1
A)
Crosstalk between PD-1 and SOCS-1 to inhibit STAT-1/5 phosphorylations
B) Control IgG a-PDL-1
pSTAT-1
Total STAT-1
- + + + Core
D) Control IgG Anti-PDL-1 Control IgG Anti-PDL-1
%S
TA
T-5
+C
D14
+ c
ells **
%S
TA
T-1
+C
D14
+ c
ells *
ST
AT
-1
CD14
ST
AT
-5
CD14
Viral persistence
gC1qR
HCV core
Th1/Tc1 dysregulation
Signaling pathways for IL-12 expression (Jak/STAT)
SOCS-1
TLRs
Viral clearance
LPS/R848PD-L1
PD-1
Our Model
Tim-3
Gal-9
What is the underlying mechanism leading to these immunodysregulations?
Immunodysregulation in chronic HCV infection
Decreased IL-12 Impaired Monocyte maturation into DC
T cell dysfunction
/ exhaustion
mixed cryoglobulinemia
non-Hodgkins lymphoma
B cell clonal expansion
Viral Persistence
B cell hyperactivation
HCV infection lead to a differential effect on T/B lymphocytes - what is the underlying mechanism ?
Why we care about this?
-Negative signaling molecules in HCV pathogenesis
TA
LL
-1
IgG
IgM
CD20 CD20 CD20
10.4% 96.2% 83.3%
4.2% 72.3% 48.8%
HCV-NHL
HS
HC
V-T
etra
mer 5.8%
19.1%
19.4%
45.3%
CD8
CD
69
CD4
HCV-NHL
HS
Cell Immunology & Biology 2011
PD
-1
HCV-NHL
CD4
47.3%
37.2%
70.0%
53.5%HCV
Tetramer CD8
HCV-NHL
HS
CD20
PD
-16.9%
13.7%
PD
-1
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
+ - + - Core
T cells B cells
SOCS-1 -Actin
+ - + - Core
T cells B cells
SOCS-1 -Actin
Differential regulation of T / B lymphocyte signaling by HCV core protein
HCV-NHL HS HCV-NHL HS
____________ ____________T cells B cells
SOCS-1
hβ2M
T cells B cells____________ ____________
HCV-NHL HS HCV-NHL HS
SOCS-1
Differential regulation of T/B lymphocyte activation in patients with HCV-NHL
pSTAT1
B) Anti-PD-L1 Control Ab
HS
HCV-NHL
80% 10% 9% 1% 46% 13% 36% 5%
22% 36% 38% 4% 1% 9% 77% 13%
CFSE
T c
ell C
ount
s
21.3% 13.7%
Anti-PD-L1 Control Ab
CD4
CD8
HC
V-T
etra
mer
A)
CD
69 29.8% 17.1%
Blocking PD-1 signaling restores T cell activation and proliferation
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
IL-23 p19
IL-1
2 p3
5
HCV HS ** *
CD4
IL-1
7A
HCV HS ** Pearson r=0.465p < 0.05
IL-23/IL-12 by CD14+ cells
STAT-1 STAT-3
TH17
IL-12
IL-23
Tim-3
TLR TLR
monocyte
Gal-9
HCV
Tim-3
monocyte
monocyte monocyte
Hepatocyte HepatocyteHepatocyte
Foxp3+ Tregs
A)
B)
**
*** *
8.91 11.94
74.75 4.40
13.54 20.85
53.68 11.93
1.54 5.68
52.82 39.95
2.44 9.41
62.70 25.45
HS HCV
HS HCV
Differential regulation of IL-12/IL-23 expressions by M/MФ leads to TH17 cell and Foxp3+ Treg development during HCV infection
CD4+ CD4+
CD25+
CD4+
CD25+
Foxp3+
CD4+
CD25+
Foxp3-
* NS ** NS
A) B)
25.86 15.78
28.50 29.85
20.06 7.42
45.08 27.44
15.94 4.89
62.47 16.7
14.53 7.26
59.18 19.03
Pearson Correlation = 0.75Sig. (1-tailed)=0.0002; (2-tailed)=0.0004
HS HCV HS HCV
Differential regulation of IL-12/IL-23 expressions by monocytes/macrophagesleads to TH17 cell and CD4+CD25+Foxp3+development during HCV infection
CD25+ FoxP3-
T eff
CD25+ FoxP3+ T reg
apoptosis proliferation
Teff
Treg
TregTreg
apoptosis proliferation
CD4+ T cell
Tim-3/Gal-9
α-Tim-3
TGF-β/IL-10
IL-2
HCV-infected hepatocytesproduce Gal-9 and TGF-β
Tim-3 is up-regulated more on Foxp3+ Tregs than on Teffs
Tim-3/Gal-9 interactions shift the balance of Tregs/Teffs by regulating T cell proliferation and apoptosis
α-Tim-3 may correct the imbalance of Tregs/Teffs ratio induced by HCV
Moorman JP et al J Immunol 2012
monocyte IL-23/IL-12
IL-17
Diminished CD4+/ CD8+ T cells
Increased IL-23
Immunodysregulation during chronic HCV infection
Aberrant CD19+ B cell activation
Decreased IL-12 Impaired CD14+ M/MΦ maturation into DC
Accumulated TH17 & Foxp3+ Treg cells
HCV-infected Hepatocytes
Increased PD-1, SOCS-1, Tim-3
Increased IL-17
Increased IL-10
Increased TGF-β
Decreased IL-2
Decreased TNF-α
Decreased IFN-γ
Increased IgG
Increased IgM
HCV chronic infection
Autoimmune disorders
Why we care about this?
-Negative signaling molecules in Vaccine response
HBV vaccine response and HCV vaccine development
Isotype
HBV-R
HBV-NR
% T
im-3
/CD
14+ c
ells
**
*
*** A)
B)
Tim-3 on HBV vaccine failure in HCV-infected individuals
HBV Vaccine response: 90% in Healthy Subjects; 50% in HCV-infected patients%
IL
-12p
35/C
D14
+ c
ells
% I
L-2
3p19
/CD
14+ c
ells
PD-1+ CD4+ T cells
CD
69+ C
D4+
T c
ells
Pearson Corr. = - 0.374**Sig.(2-tailed) = 0.001
HBV-R HBV-NR HBV-R HBV-NR
SOCS-1
β-actin
HBsAg stimulation a-CD3/28 stimulation
0
0.1
0.2
0.3
0.4
SO
CS-
1/ac
tin
HBsAg stimulation a-CD3/28 stimulation
HBV-R HBV-NR HBV-R HBV-NR
*
*
PD-1/SOCS-1 on HBV vaccine failure in HCV-infected individuals
HCV patientsHBV-NR (n=29)
HCV patientsHBV-R (n=32)
HCV resolvedindividuals(n=6)
Healthy Subjects(n=10)
P
D-1
exp
ress
ion
on
CD
4+ T
cel
ls 12.1% vs 7.0%, P=0.002 5.6% vs 4.5%, P>0.05
9.4% vs 4.9%, P=0.007
A)29.9%18.1%
1.5% 8% 18% 73% 0% 0.3% 7% 92%
0% 1.5% 19% 79% 1% 21% 58% 20%
57.8%31.6%
Control Ab a-PD-L1 Ab
HBsAg stimulation
a-CD3/28 stimulation
B)
HBsAg stimulation
a-CD3/28 stimulation
Control Ab a-PD-L1 Ab
HBsAg stimulation a-CD3/28 stimulation
%
CD
69+
in C
D4+
T c
ells
*
*
**
* *
*
IgG
a-PDL-1
IgG
a-PDL-1
IgG
a-PDL-1
M1 M2 M3 M4
M1 M2 M3 M4
C
FS
E /
HB
sAg
CF
SE
/ a
-CD
3/28
Monocyte
iDC induced by GM-CSF + IL-4
mDC induced by TNF-α + Poly I:C
mDC infected by Lm-HCV vaccine
CD14 HLA-ABC HLA-DR CD209 CD1a CD80 CD83 CD86
Lm-NS5BLm-infected DC
Why do we care - Listeria monocytogenes (Lm)-based DC-targeting HCV vaccine DevelopmentHCV vaccine development: HCV-quasispecies; HCV-delivery; HCV-models; HCV-exhaustion
Lm vector NS5B ∆actA/∆inlB
Listeria monocytogenes
HCVantigens
Virulence determinants
%T
im-3
+ c
ells
Why do we care ? Improve Lm-based DC-targeting HCV vaccine by blocking Tim-3 signaling
%IL
-12
+ cel
ls
Un-infected HCV HCV+IgG HCV+a-Tim-3
M/MФ
iDC
mDC
BSA-
FITC
Upt
ake
(ΔM
FI)
IgG α-Tim-3
Lm-control Lm-NS5B IgG α-Tim-3
CD3+CD8+
B)
CD3+CD8+
Iso HCV-resolved HCV-infected
Tim-3
A)Gating strategy
Why do we care?Improve Lm-based DC-targeting HCV vaccine by blocking Tim-3 signaling
Granzyme-B
IFN-γ
A)
B)
Why do we care?Listeria monocytogenes (Lm)-based DC-targeting HCV vaccine
HCV persistence
PD-1CD28CD3 gC1qR
HCV Core PD-L1 MHC/peptide/B7
T cell dysfunction
T cell activation α-PD-1
α-Tim-3
α-gC1qR
α-HCV core
viral clearance
SOCS
Negative T cell regulators
LPS
TLR
STAT
Monocyte IL-12
Tim-3
Gal-9
Why do we care?- novel therapeutics
Improve HBV vaccine response in HCV/HIV-infected individuals
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