PD-1, SOCS-1, Tim-3 in HCV infection
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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.

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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
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

  • 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


200 M WW

4 M U.S.

Clinical features of HCV infection

15%

PD-1, SOCS-1, Tim-3

HIV

Why the majority of infected individuals become chronic?


B cell hyperactivation

mixed cryoglobulinemia

T cell dysfunction

and exhaustion

non-Hodgkins lymphoma

Immunodysregulation in chronic HCV infection

Impaired Monocyte maturation into DC

Decreased IL-12

Viral Persistence

B cell clonal expansion

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

SOCS-1

HCV+ Huh-7 HCV- Huh-7

PD-1

Tim-3

So HCV-infected hepatocytes has ample opportunity to contact circulating or infiltrating immune cells

Tim-3+ CD14+ M/MØ

6 h 12 h 24 h 48 h


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

What is PD-1



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

15.2%

Gating strategy

CD14

IL-12

60%

**

__________________________

50%

*

____________

**

____________________________

40%

PD-1+ CD14+ cells

60%

*

_______________

30%

50%

20%

40%

10%

30%

IL-12+ CD14+ cells

0%

20%

HCV-InfectedHCV-Resolved Healthy

10%

0%

HCV-InfectedHCV-Resolved Healthy

Ma et al. Immunology 2010; Zhang et al. J Immunol 2011


PD-1 is inversely associated with IL-12 production by monocytes

B)

Pearson Correlation = -0.464*

A)

LPS/R848

PD-1

PD-1+ CD14+ cells

IL-12

IL-12 production

C)

D)

50%

50%

P = 0.003

P = 0.034

40%

40%

PD-1+ CD14+ Cells

IL-12+ CD14+ Cells

30%

30%

20%

20%

10%

10%

0

0

before after

IFN/RBV IFN/RBV

before after

IFN/RBV IFN/RBV


Tim-3 is a negative molecule expressed on resting monocytesmonocytes

to control IL-12 expression

Positive cells (%)

isoun-stimulatedTLR-stimulated

isoun-stimulatedTLR-stimulated

2.7%

0.2%

1.8%

Tim-3

55.3%

0.4%

2.2%

Tim-3

1.2%

3.0%

45.8%

0.1%

0.1%

38.9%

Time of TLR stimulation

IL-12

CD14

CD14

0.3%

0.2%

27.2%

IL-12

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 HCV Patient

Isotype control Naïve Activated Naïve Activated

2.8

4.1

4.4

54.6

3.4

8.7

7.4

38.3

Tim-3

47.6

18.8

74.4

45.5

22.3

13.5

27.3

27.0

CD14

40.9

33.9

54.0

15.3

71.3

0.2

67.7

0.4

CD14

2.5

1.0

0.1

22.7

0.2

29.6

28.5

31.8

IL-12

**

***

B

C

NS

**

***

***

***

***

% of IL12+ CD14+ M/MØ

% of Tim-3+ CD14+ M/MØ

Healthy Subjects Chronic HCV Patients

Naïve Activated Naïve Activated

Naïve Activated 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 HCV patients

B)

Healthy Subjects HCV patients

99.5%

10.3%

40.7%

99.7%

*

TLR7+ CD14+ Cells

TLR4+ CD14+ Cells

Healthy Subjects HCV patients

Healthy Subjects HCV patients

C)

Healthy subject HCV-infected HCV-resolved

STAT-1+ CD14+ Cells

21.6%

9.6%

23.7%

*

Healthy HCV-infected HCV-resolved

D)

IgG anti-Tim-3

+ + Core

Phospho Stat1

Total Stat1


gC1qR intracellular signaling

PD-1

Isotype 1.2%

LPS R848 11.7%

LPS R848+core+IgG 3.2%

LPS R848+core+a-PDL-1 8.9%

Control IgG a-PDL-1

+ + Core

SOCS-1

β-actin

HCV core

TLR

Count

* *

M/MФ IL-12 production

SOCS-1

% IL-12+CD14+ cells

* *

IL-12


Silencing SOCS-1 inhibits PD-1 expression and improve IL-12 production

A)

+ + + + Core

Isotype 0.73%

LPS R848 Core control siRNA 39.1%

Isotype 2.88%

48 h after transfection72 h after transfection

Control siRNASOCS-1 siRNAControl siRNASOCS-1 siRNA

LPS R848 Core SOCS-1 siRNA 2.9%

LPS R848 Core control siRNA 10.1%

LPS R848 Core SOCS-1 siRNA 19.4%

B)

SOCS-1

Count

β-Actin

PD-1

C)

Count

IL-12


Crosstalk between PD-1 and SOCS-1 to inhibit STAT-1/5 phosphorylations

A)

B)

Control IgG a-PDL-1

Control siRNA SOCS-1 siRNA

+ + Core

- + + + Core

pSTAT-1

pSTAT-1

Total STAT-1

Total STAT-1

D)

Control IgG Anti-PDL-1

Control IgG Anti-PDL-1

STAT-1

STAT-5

CD14

CD14

**

*

%STAT-5+CD14+ cells

%STAT-1+CD14+ cells


gC1qR phosphorylations

Our Model

LPS/R848

Gal-9

HCV core

PD-L1

Tim-3

PD-1

TLRs

Signaling pathways for

IL-12 expression (Jak/STAT)

SOCS-1

Th1/Tc1 dysregulation

Viral persistence

Viral clearance


B cell hyperactivation phosphorylations

mixed cryoglobulinemia

T cell dysfunction

/ exhaustion

non-Hodgkins lymphoma

Viral Persistence

Immunodysregulation in chronic HCV infection

Impaired Monocyte maturation into DC

Decreased IL-12

B cell clonal expansion

What is the underlying mechanism leading to these immunodysregulations?


Why we care about this? phosphorylations

-Negative signaling molecules in HCV pathogenesis

HCV infection lead to a differential effect on T/B lymphocytes - what is the underlying mechanism ?


CD20 phosphorylations

CD20

CD20

CD4

CD8

19.4%

45.3%

Cell Immunology & Biology 2011

5.8%

10.4%

96.2%

83.3%

IgM

IgG

TALL-1

HCV-NHL

CD69

HCV-NHL

HCV-Tetramer

19.1%

4.2%

72.3%

48.8%

HS

HS

70.0%

6.9%

HCV-NHL

HCV-NHL

PD-1

PD-1

PD-1

47.3%

37.2%

53.5%

13.7%

HCV

HS

CD4

Tetramer CD8

CD20


T cells B cells phosphorylations

+ - + - Core

SOCS-1

-Actin

Differential regulation of T/B lymphocyte activation in patients with HCV-NHL

Differential regulation of T / B lymphocyte signaling by HCV core protein

T cells B cells

T cells B cells

T cells B cells

____________

____________

____________

____________

+ - + - Core

HCV-NHL HS HCV-NHL HS

HCV-NHL HS HCV-NHL HS

SOCS-1

SOCS-1

pSTAT1

SOCS-1

-Actin

hβ2M

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

A)

B)

Anti-PD-L1

Control Ab

Anti-PD-L1

Control Ab

29.8%

17.1%

80%

10%

9%

1%

46%

13%

36%

5%

CD69

HS

T cell Counts

CD4

22%

36%

38%

4%

1%

9%

77%

13%

13.7%

21.3%

HCV-NHL

HCV-Tetramer

CFSE

CD8


Why we care about this? proliferation

-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/M proliferationФ

leads to TH17 cell development during HCV infection

HCV

HS

**

IL-17A

HCV HS

CD4

IL-12 p35

IL-23 p19

**

*

Pearson r=0.465

p<0.05

IL-23/IL-12 by CD14+ cells


Hepatocyte proliferation

Hepatocyte

Hepatocyte

TLR

TLR

monocyte

monocyte

monocyte

monocyte

 Tim-3

STAT-3

STAT-1

 IL-23

 IL-12

HCV

 Foxp3+ Tregs

Tim-3

 TH17

Gal-9


Differential regulation of IL-12/IL-23 expressions by M/M proliferationФ leads to

TH17 cell and Foxp3+ Treg development during HCV infection

HS

HCV

A)

**

11.94

20.85

8.91

13.54

11.93

74.75

53.68

4.40

B)

HS

HCV

***

*

5.68

9.41

1.54

2.44

39.95

25.45

52.82

62.70


A) proliferation

B)

HS

HCV

HS

HCV

15.78

7.42

4.89

25.86

20.06

15.94

7.26

14.53

29.85

27.44

16.7

28.50

45.08

62.47

19.03

59.18

NS

NS

*

**

Pearson Correlation = 0.75

Sig. (1-tailed)=0.0002; (2-tailed)=0.0004

CD4+

CD4+

CD25+

CD4+

CD25+

Foxp3+

CD4+

CD25+

Foxp3-


Differential regulation of IL-12/IL-23 expressions by proliferationmonocytes/macrophages

leads to TH17 cell and CD4+CD25+Foxp3+development during HCV infection

HCV-infected hepatocytes

produce Gal-9 and TGF-β

CD4+ T cell

 IL-23/IL-12

CD25+ FoxP3- T eff

 IL-17

CD25+ FoxP3+ T reg

Tim-3 is up-regulated more

on Foxp3+ Tregs than on Teffs

TGF-β/IL-10

monocyte

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

Treg

α-Tim-3

α-Tim-3 may correct the imbalance

of Tregs/Teffs ratio induced by HCV

Tim-3

/Gal-9

Moorman JP et al J Immunol 2012


Immunodysregulation during chronic HCV infection proliferation

HCV-infected Hepatocytes

Increased PD-1, SOCS-1, Tim-3

Decreased IL-12

Impaired CD14+ M/MΦ maturation into DC

Increased IL-23

Increased IL-17

HCV chronic infection

Accumulated TH17 & Foxp3+ Treg cells

Increased IL-10

Increased TGF-β

Autoimmune disorders

Decreased IL-2

Diminished CD4+/ CD8+ T cells

Decreased TNF-α

Decreased IFN-γ

Increased IgG

Aberrant CD19+ B cell activation

Increased IgM


Why we care about this? proliferation

-Negative signaling molecules in Vaccine response

HBV vaccine response and HCV vaccine development


Tim-3 on HBV vaccine failure in HCV-infected proliferationindividuals

HBV Vaccine response: 90% in Healthy Subjects; 50% in HCV-infected patients

A)

***

HBV-R

**

HBV-NR

Isotype

*

B)

% Tim-3/CD14+ cells

% IL-23p19/CD14+ cells

% IL-12p35/CD14+ cells


PD-1/SOCS-1 on HBV vaccine failure in HCV-infected individuals

9.4% vs 4.9%, P=0.007

12.1% vs 7.0%, P=0.002

5.6% vs 4.5%, P>0.05

Pearson Corr. = - 0.374**

Sig.(2-tailed) = 0.001

PD-1 expression on CD4+ T cells

CD69+ CD4+ T cells

PD-1+ CD4+ T cells

HCV patients

HBV-NR (n=29)

HCV patients

HBV-R (n=32)

HCV resolved

individuals(n=6)

Healthy

Subjects(n=10)

HBsAg stimulation a-CD3/28 stimulation

*

HBV-R HBV-NR HBV-R HBV-NR

*

SOCS-1/actin

SOCS-1

β-actin

HBV-R HBV-NR HBV-R HBV-NR

HBsAg stimulation a-CD3/28 stimulation


Control Ab a-PD-L1 Ab

A)

IgG

*

18.1%

29.9%

a-PDL-1

HBsAg stimulation

*

% CD69+ in CD4+ T cells

31.6%

57.8%

HBsAg stimulation a-CD3/28 stimulation

a-CD3/28 stimulation

IgG

Control Ab a-PD-L1 Ab

B)

a-PDL-1

0% 0.3% 7% 92%

1.5% 8% 18% 73%

CFSE / HBsAg

*

HBsAg stimulation

*

M1 M2 M3 M4

IgG

1% 21% 58% 20%

0% 1.5% 19% 79%

*

a-PDL-1

a-CD3/28 stimulation

*

*

CFSE / a-CD3/28

M1 M2 M3 M4


Why do we a-PD-L1 Abcare - Listeriamonocytogenes (Lm)-based DC-targeting HCV vaccine Development

HCV vaccine development: HCV-quasispecies; HCV-delivery; HCV-models; HCV-exhaustion

Listeria

monocytogenes

HCV

antigens

Virulence

determinants

Lm-NS5B

∆actA/∆inlB

Lm vector

NS5B

Lm-infected DC


Why do we a-PD-L1 Abcare?

Improve Lm-based DC-targeting HCV vaccine by blocking Tim-3 signaling

M/MФ

BSA-FITC Uptake (ΔMFI)

iDC

%Tim-3+ cells

%IL-12 + cells

mDC

Un-infected HCV HCV+IgG HCV+a-Tim-3


Why do we care? a-PD-L1 Ab

Improve Lm-based DC-targeting HCV vaccine by blocking Tim-3 signaling

%HCV-Tet+/CD3+CD8+

HCV-Tetramer

%Tim-3+/HCV-Tet+CD3+CD8+

Lm-control

Lm-NS5B

SSC

HCV-Tetramer

α-Tim-3

IgG

IgG

α-Tim-3

A)

Gating strategy

CD3+CD8+

Tim-3

CD3+CD8+

B)

Iso

HCV-resolved

HCV-infected


Why do we care? a-PD-L1 Ab

Listeria monocytogenes (Lm)-based DC-targeting HCV vaccine

A)

%IFN-r+/HCV-Tet+CD3+CD8+

IFN-γ

B)

%GranzymeB+/HCV-Tet+CD3+CD8+

Granzyme-B


Why do we care?- a-PD-L1 Abnovel therapeutics

HCV Core

MHC/peptide/B7

PD-L1

LPS

Gal-9

gC1qR

TLR

PD-1

CD3

CD28

Tim-3

STAT

Monocyte IL-12

α-gC1qR

α-HCV core

SOCS

T cell activation

α-PD-1

α-Tim-3

Negative T cell regulators

Improve HBV vaccine response

in HCV/HIV-infected individuals

T cell dysfunction

viral clearance

Improve HCV - DC

therapeutic Vaccine

HCV persistence


Acknowledgements a-PD-L1 Ab

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


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