Feras Al-Ghazawi, Parmvir Parmar, Elliott Faller and Paul MacPherson

University of Ottawa and The Ottawa Hospital Research Institute

Suppressor of cytokine signaling (SOCS) proteins are induced by IL-7 and the interaction of SOCS proteins with the IL-7 receptor alpha (CD127) may play a role in regulating CD127 expression in human CD8 T cells

2

Proliferation

Apoptosis

Survival

Apoptosis

+ IL-7

CD8 T cell

Clonal Expansion

Memory

Cytotoxic molecules

CTL response

MHC I

Ag

TCR

+ IL-7

Clonal Expansion

Memory

CTL response

Infected/cancer cell

Memory

Homeostatic response

Antigen-specific CTL response

3

IL-7 Receptor

http://en.wikipedia.org

• Heterodimer composed of a unique α-chain (CD127) and a common γ-chain (CD132) that is shared with the receptors for IL-2, -4, -9, -15 and -21.

• IL-7Rα (CD127) is a 75 kDa type-I transmembrane protein with an extracellular domain and a cytoplasmic tail.

• IL-7Rα is majorly expressed on T cells and their precursors.

4

IL-7 signaling is dysregulated in HIV infection

• HIV infected individuals have increased plasma concentrations of IL-7 averaging 18 pg/ml compared to the much lower average 2.2 pg/ml found in the sera of HIV-negative controls.

• Surface expression of CD127 is down regulated on CD8 T-cells in HIV infected patients and is partially restored in patients receiving HAART.

• IL-7 is currently being examined in clinical trials to increase T cell numbers in lymphopenic patients due to BMT, chemotherapy and HIV infection.

• Therefore, it is important to understand how does IL-7 regulate the expression of its own receptor CD127.

5

CD127

M 3 6 12 24

Hsp70

CD127 N=6

Med

ia 3h 6h 12h

24h

0.0

0.5

1.0

1.5

*

**

IL-7 10ng/ml

Rel

ativ

e P

rote

in E

xpre

ssio

n

Hours

IL-7, 10 ng/ml

IL-7 Down Regulates Total CD127 Protein

N=6IL-7=10 ng/ml

6

CD127

β actin

CD127 N=8

Med

iaIL

-7

MG

132

MG

132+

IL-7

0.0

0.5

1.0

1.5

*

Rel

ativ

e P

rote

in E

xpre

ssio

n

M IL-7 MG MG+IL-7

N=8IL-7=10 ng/mlMG132=5 µM t=6hrs

IL-7-mediated CD127 Down regulation is Dependent on 26S Proteasome

7

Suppressor of cytokine signalling (SOCS) proteins

• Cytokine-inducible negative regulators of cytokine signaling.

• Eight members (SOCS1-7 and CIS).

• Common SH2 domain (interacts with phospho. machinery), C-term SOCS box (recruits E3 unbiquitin ligases), N-term. of varying lengths.

Akhtar LN, Benveniste EN.J Virol. 2011 Mar;85(5):1912-21

8

CIS

SOCS1

SOCS2

SOCS3

SOCS4

SOCS5

SOCS6

SOCS7

0.0

0.5

1.0

10

20

30

Rel

ativ

e m

RN

A e

xpre

ssio

n

Basal levels – unstimulated CD8 T cells

SOCS1-7 and CIS transcripts are expressed in human CD8 T cells

N=5

9

SOCS1

0 10 20 30 40 500

5

10

15

20

25

0.1 ng ml

Media10 ng/ml1 ng/ml

Time (hr)

Re

lati

ve

SO

CS

1 m

RN

A e

xp

res

sio

n

SOCS2

0 10 20 30 40 500

100

200

300

400Media10 ng/ml1 ng/ml

0.1 ng ml

Time (hr)

Re

lati

ve

SO

CS

2 m

RN

A e

xp

res

sio

n

CIS

0 10 20 30 40 500

5

10

15

0.1 ng ml

Media10 ng/ml1 ng/ml

Time (hr)

Re

lati

ve

CIS

mR

NA

ex

pre

ss

ion

IL-7 induces the expression of CIS/SOCS1-2 mRNA transcripts in CD8 T cells

N=3

10

SOCS1

MIL

-7JA

Ki

JAKi+

IL-7

PI3Ki

PI3Ki+

IL-7

0

5

10

15

20

25

mR

NA

exp

ress

ion

(fo

ld)

SOCS2

MIL

-7JA

Ki

JAKi+

IL-7

PI3Ki

PI3Ki+

IL-7

0

100

200

300

mR

NA

exp

ress

ion

(fo

ld) CIS

MIL

-7JA

Ki

JAKi+

IL-7

PI3Ki

PI3Ki+

IL-7

0

5

10

15

mR

NA

exp

ress

ion

(fo

ld)

IL-7 mediated induction of SOCS mRNA is JAK-dependent

N=3IL-7=10 ng/mlJAKi=1.5 µMPI3Ki=50 µMt=3hrs

11

SOCS1

MIL

-7

STAT5i

STAT5i

+IL-7

0

10

20

30

mR

NA

exp

ress

ion

(fo

ld)

SOCS2

MIL

-7

STAT5i

STAT5i

+IL-7

0

100

200

300

mR

NA

exp

ress

ion

(fo

ld)

CIS

MIL

-7

STAT5i

STAT5i

+IL-7

0

5

10

15

mR

NA

exp

ress

ion

(fo

ld)

IL-7 mediated induction of SOCS mRNA is STAT5-dependent

N=3IL-7=10 ng/mlSTAT5i=0.5 mMt=3hrs

12

IL-7 mediated induction of SOCS mRNA does not depend on new protein synthesis

N=4IL-7=10 ng/mlCHX=100 µMt=1hr

SOCS1

MIL

-7CHX

CHX+IL-7

0

2

4

6

8

Rel

ativ

e m

RN

A E

xpre

ssio

n SOCS2

MIL

-7CHX

CHX+IL-7

0

10

20

30

40

50

Rel

ativ

e m

RN

A E

xpre

ssio

n CIS

MIL

-7CHX

CHX+IL-7

0

5

10

15

Rel

ativ

e m

RN

A E

xpre

ssio

n

13

SOCS2

β actin

M 30’ 1h 2h 3h 6h 12h 24h 48h

SOCS1

CIS

β actin

β actin

SOCS1

M 30' 1 2 3 6 12 24 48

0

1

2

3

4

5

Time (hr)

Re

lati

ve

Pro

tein

Ex

pre

ss

ion

CIS

M 30' 1 2 3 6 12 24 48

0

1

2

3

4

5

Time (hr)

Re

lati

ve

Pro

tein

Ex

pre

ss

ion

SOCS2

M 30' 1 2 3 6 12 24 48

0.0

0.5

1.0

1.5

2.0

Time (hr)

Re

lati

ve

Pro

tein

Ex

pre

ss

ion

IL-7 induces the expression of CIS and SOCS1-2 proteins

N=4IL-7=10 ng/ml

15

SOCS1

SOCS2

CIS

Media - IP: G

oat anti-CD127 (M)

IL-7 3h - IP: Goat anti-CD127 (IL-7)

Media - IP: G

oat Isotype

CIS, SOCS1 and 2 proteins physically bind to CD127 in CD8 T cells

N=5IL-7=10 ng/ml

16

SOCS1 Med

iaM

edia

IL-7

IL-7

SOCS2

AM

edia

IL-7

CIS

CID127 co-localization with: B

SOCS1 Med

iaM

edia

IL-7

IL-7

SOCS2

Med

iaIL

-7

CIS

EEA1 co-localization with:

IL-7 induces co-localization of CIS and SOCS2 with CD127 and with the early endosome marker EEA1

17

C

SOCS1 Med

iaM

edia

IL-7

IL-7

SOCS2

Med

iaIL

-7

CIS

LAMP1 co-localization with:

SOCS1 Med

iaM

edia

IL-7

IL-7

SOCS2

D

Med

iaIL

-7

CIS

MCP20 co-localization with:

CIS and SOCS2 co-localize with CD127 and with markers of the proteasome but not with markers of the lysosome upon IL-7 stimulation

18

SOCS2

β actin

CD127

M IL-7

SMER3

SMER3+IL7

CIS

The degradation of CD127 and SOCS proteins are dependent on E3 ubiquitin ligases

N=3IL-7=10 ng/mlSMER3=30M

SOCS2

CIS

SOCS1

A

β actin

β actin

β actin

siRNAαSOCS2M

0.0

0.50.5

0.6

0.7

0.8

0.9

1.0 **

NS*

*NSNS

NS

Rela

tive

surf

ace

CD

127

exp

ressi

on

* *

B

Contribution of SOCS1, SOCS2 and CIS proteins in the IL-7 mediated down regulation of CD127 as measured by siRNA-mediated knock-down assays

siRNAαCISM

siRNAαSOCS1

M

N=4IL-7=10 ng/ml

20

PP

IL-7 binding

CD127 CD132

STAT5

JAK3JAK1

CD127

(B) IL-7 Stimulation(A) Resting CD8 T-Cells

CD127

Homeostatic Recycling

Lysosomal Degradation

Proteosomal Degradationof SOCS-CD127 complexes

Internalization andphosphorylation of CD127

STA

T5

CIS

P

SOCS2

CIS and SOCS2proteins

CD127 protein

Early Endosome

P

1- binding of CIS and SOCS2 to phosphorylated CD1272- Polyubiquitination of SOCS box by E3 ubiquitin ligases

P

STA

T5 P

STAT5PSTAT5

P

STAT5PSTAT5

P

21

Acknowledgements

• Blood donors

22

A

Med

iaIL

-7 Jaki

Jaki

+IL-

7

pY449-CD127

CD127

C

B

CD127

β actin

Med

ia

IL-7Jaki

Jaki

+IL-

7

0 3 6 9 12 15 18 21 240.0

0.2

0.4

0.6

0.8

1.0

1.2MIL-7JakiJaki + IL-7

Time (hr)Rel

ativ

e su

rfac

e C

D12

7 ex

pre

ssio

n