pd-1, socs-1, tim-3 in hcv infection -why we care? yao, z. q. m.d. ph.d. director, hepatitis...

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



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%

*



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?



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 ?



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



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


-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


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


%

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

pd-1, socs-1, tim-3 in hcv infection -why we care? yao, z. q. m.d. ph.d. director, hepatitis...

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