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Modulatory signalling in immune cells: SLAM family receptors and SAP-related adaptors André Veillette 414A November 2004. Fc g RIII (NK cells). TCR (T-cells). Fc e RI (mast cells). a. gg. gg. ge. de. b. zz. Immunoreceptors. NCRs NKG2D (NK cells). BCR (B-cells). DAP-12.

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slide1

Modulatory signalling in immune cells:

SLAM family receptors and

SAP-related adaptors

André Veillette

414A

November 2004

slide2

FcgRIII

(NK cells)

TCR

(T-cells)

FceRI

(mast cells)

a

gg

gg

ge

de

b

zz

Immunoreceptors

NCRs

NKG2D

(NK cells)

BCR

(B-cells)

DAP-12

Iga/Igb

= ITAM

slide3

PH

TCR signalling

MHC

CD4

TCR

CD4

TCR

Ag/MHC

P

P

P

P

P

P

ge

P

P

de

SH2

zz

SH2

PH

P

P

Lck

P

FynT

FynT

SH2

Lck

SH2

Itk

Zap-70

Zap-70

protein tyrosine phosphorylation

Itk

slide4

T-cell signalling

Modulatory

signalling

Positive

signalling

Inhibitory

signalling

PAG

(ITIMs)

Csk-PEP

SHP-1

c-Cbl

TCR-CD4/CD8

Src kinases

Zap-70

Btk kinases

CD28

PI 3’ kinase

ICOS

?

SLAM

?

SAP

? ? ? ? ? ? ?

Cytokine

production

(TH1 vs TH2)

Anergy

Maturation

Memory

Apoptosis

slide5

SAP family of adaptors

SH2

SAP

(T-cells, NK cells, NK-T-cells, ?some B-cells)

EAT-2

(NK cells, macrophages,

DCs, ?some B-cells)

SH2

slide6

X-linked lymphoproliferative disease (XLP)

  • - caused by inactivating point mutations or deletions in the sap gene;
  • characterized by abnormal response to EBV infection, resulting in fatal infectious mononucleosis, as well as a high frequency
      • of malignant lymphomas;
  • - SAP-deficient mice exhibit abnormal CD4+ T-cell help (decreased TH2 functions; +/- increased TH1 functions), decreased Ig production and memory B-cell generation, altered anti-viral responses and absent NK-T-cells.
slide7

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

SAP family adaptors associate

with SLAM-related receptors

NTB-A

Ly-9

CRACC

CD48

CD84

SLAM

measles

Ligands

Ly-9

V

2B4

CRACC

SLAM

NTB-A

CD84

C2

V

V

V

V

V

V

C2

C2

C2

C2

C2

C2

Y

Y

Y

Y

: TIYxxV/Imotif

Y

Y

Y

Y

Y

Y

Y

: TVYxxV/Imotif

Y

Y

Y

Y

Y

Y

slide9

XLP is likely caused by multiple

SLAM-related receptor dysfunctions

Ig production

memory B-cells

?

TH2

functions

NK cell

cytotoxicity

?

SLAM

NTB-A

CD84

?

SAP

Ly-9

2B4

NK cell

cytotoxicity

slide11

SLAM (CD150)

  • expressed on activated T-cells, B-cells,
      • macrophages and dendritic cells;
  • self-ligand;
  • SLAM is the lymphoid-specific receptor
  • for measles virus in humans;
  • - anti-SLAM stimulation modulates IFN-g secretion
  • by activated T-cells;
  • defective TCR-induced IL-4 production in
  • slam-/-mice.
slide12

SAP is required for SLAM-induced

protein tyrosine phosphorylation

slide13

PH

-

-

?Ras

?

?Itk

?Akt

SLAM

MHC

CD4

TCR

SLAM

SH2

SAP

P

P

P

P

P

P

Lck

P

P

P

P

SH2

SH2

P

P

kinase

P

FynT

Zap-70

Ras-GAP

Dok

SHIP-1

Itk

IL-4 production

slide14

Possible mechanism(s) by which SAP

induces protein tyrosine phosphorylation:

1) displacement of a protein tyrosine phosphatase;

2) recruitment of a protein tyrosine kinase;

3) both.

slide15

A

- SAP

ligand

SLAM

+ ligand

SH2

SH2

SH2

SH2

SHP-2

SHP-2

tyrosine phosphorylation

B

+ SAP

ligand

SLAM

+ ligand

SAP

SAP

SH2

SH2

P

P

tyrosine phosphorylation

SAP: a natural blocker of SH2 domain-mediated interactions?

slide18

SLAM

MHC

CD4

TCR

SLAM

SH2

SAP

P

P

P

P

P

P

Lck

P

P

P

P

SH2

SH2

P

P

FynT

PH

P

FynT

Zap-70

Itk

How does SAP promote recruitment of FynT?

SH3

SH2

slide19

SAP is a bifunctional adaptor that

links SLAM to FynT

SAP

SLAM

FynT

SH2

pY

SH3

SH3

P

P

P

P

slide20

The FynT SH3 domain binds a novel arginine-based

motif at the surface of the SAP SH2 domain

SH2 domain

1 30

SAP(m)MDAVTVYHGKISRETGEKLLLATGLDGSYLLRDSESVPGVYCLCVLYQGYIYTYRVSQTETGSWSAE

SAP(h)MDAVAVYHGKISRETGEKLLLATGLDGSYLLRDSESVPGVYCLCVLYHGYIYTYRVSQTETGSWSAE

EAT-2(m)MD.LPYYHGCLTKRECEALLLKGGVDGNFLIRDSESVPGALCLCVSFKKLVYSYRIFREKHGYYRIE

EAT-2(h)MD.LPYYHGRLTKQDCETLLLKEGVDGNFLLRDSESIPGVLCLCVSFKNIVYTYRIFREKHGYYRIQ

68 126

SAP(m)TAPGVHKRFFRKVKNLISAFQKPDQGIVTPLQYPVE.KSSGRGPQAPTG.RRDSDICLNAP

SAP(h)TAPGVHKRYFRKIKNLISAFQKPDQGIVIPLQYPVEKKSSARSTQGTTGIREDPDVCLKAP

EAT-2(m)TDAHTPRTIFPNLQELVSKYGKPGQGLVVHLSNPIMRNNLC...QRGRRMELELNVYENTDEEYVDVLP

EAT-2(h)TAEGSPKQVFPSLKELISKFEKPNQGMVVHLLKPIKRTSPS...LRWRGLKLELETFVNSNSDYVDVLP

A

1

B

C

D

E

tail

*

*

*

*

*

*

*

F

2

G

slide23

SH3

SH3

P

P

P

P

P

A common mechanism for signalling

by SLAM-related receptors

SLAM

CD48

SLAM

2B4

Y

Y

SH2

SH2

Y

SAP

SAP

P

Y

Y

P

P

Y

FynT

FynT

Y

SHIP-1

Dok-1/2

Ras-GAP

Vav-1

SHIP-1

c-Cbl

Modulation

of IL-4

Modulation of cytotoxicity

Modulation of IFN-g

slide24

Role of FynT

in SAP-dependent signalling in vivo?

slide25

fyn-/- T-cells exhibit TH2 and, to a lesser extent,

TH1 cytokine defects in response to TCR stimulation

slide26

Creation of a sapR78A “knock-in” mouse

floxed sapR78A allele

*

*

cgggcg

TGA

ATG

sapR78A

loxP

exon 1

exon 2

exon 3

exon 4

R78A

SH2 domain

slide27

sap+

sap+

sap-

sap-

sapR78A

sapR78A

Anti-CD3:

Anti-CD28:

0.3

1

3.0

0

0.3

0

3.0

1

0

0

P+I

Cytokine production defect in T-cells from sapR78A mice

IFN-g

IL-4

2500

400

350

2000

300

IFN-g (pg.ml-1)

250

1500

IL-4 (pg.ml-1)

200

1000

150

100

500

50

0

0

Anti-CD3:

Anti-CD28:

0.3

1

3.0

0

0.3

0

3.0

1

P+I

0

0

IL-13

Proliferation

300000

300

250000

250

cpm

200000

200

IL-13 (pg.ml-1)

150000

150

100000

100

50000

50

0

0

Anti-CD3:

Anti-CD28:

Anti-CD3:

Anti-CD28:

0.3

1

0.3

1

3.0

0

3.0

0

0.3

0

3.0

1

P+I

0.3

0

3.0

1

0

0

0

0

P+I

slide28

Defective IgE production in sapR78A mice

A

B

9

8

10

7

sap+

6

day 0

8

sap-

day 14

5

IgE (mg.ml-1)

sapR78A

6

4

IgE (mg.ml-1)

4

3

2

2

1

0

0

0

14

28

35

42

49

70

sapR78A

sap+

sap-

time (days)

C

D

40

35

35

30

30

day 0

25

25

day 14

20

IgE (mg.ml-1)

20

IgG1 (mg.ml-1)

15

15

10

10

5

5

0

0

sap-

sapR78A

control

fyn-/-

sap-

sapR78A

control

fyn-/-

slide29

DC

?

MHC

TCR

?

T-cell

SAP

FynT

FynT

IL-4

IL-13

Regulation of TH2 cytokine production by SAP-FynT pathway

slide32

Regulation of TH2 cytokine production by

the SLAM-SAP-FynT pathway

DC

MHC

MHC

SLAM

SLAM

TCR

TCR

SLAM

T-cell

SAP

SAP

FynT

FynT

FynT

FynT

P

GATA-3

IFN-g

IL-4

IL-13

slide33

Conclusions

1) SAP, a small SH2 domain-containing adaptor

mutated in XLP, is necessary for modulatory

signalling through SLAM-related receptors;

2) SAP functions by recruiting the Src-related PTK

FynT, rather than by displacing a PTP;

3) SAP links SLAM-related receptors to FynT

through binding of a unique surface in

the SAP SH2 domain to the FynT SH3 domain;

4) Defects in SAP-mediated recruitment of FynT

to SLAM receptors are likely to underlie

at least part of the pathophysiology of XLP.

slide34

Riyan Chen

Romain Roncagalli

Oliver Utting

James Taylor

Laurent Doucet

Ming-Chao Zhong

Mario-Ernesto Cruz-Munoz

Marceline Côté

Xiaochu Shi

Shaohua Zhang

Dominique Davidson

Sylvain Latour

Collaborators:

Pam Schwartzberg

Rusung Tan

Yusuke Yanagi

Luo Yin

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