Modulatory signalling in immune cells:SLAM family receptors and

SAP-related adaptors

André Veillette414A

November 2004

TCR(T-cells)

Ig/Ig

BCR(B-cells)

FcRIII(NK cells)

FcRI(mast cells)

= ITAM

DAP-12

NCRs NKG2D

(NK cells)

Immunoreceptors

TCR signalling

Lck FynTSH2

SH2

Zap-70

CD4 TCR

SH2

SH2

Lck

Zap-70

CD4 TCR

FynT

P

PP

P

PP PP P

PP

Ag/MHC

MHC

protein tyrosine phosphorylation

PH

Itk

PH

Itk

PAG(ITIMs)

Csk-PEPSHP-1c-Cbl

TCR-CD4/CD8

Src kinasesZap-70

Btk kinases

T-cell signalling

Positivesignalling

Inhibitorysignalling

Cytokineproduction

(TH1 vs TH2)

AnergyMemoryMaturation Apoptosis

SAP

Modulatorysignalling

CD28

PI 3’ kinase

ICOS

?

SLAM

?

? ? ? ? ? ? ?

SH2 SAP(T-cells, NK cells, NK-T-cells, ?some B-cells)

SH2 EAT-2 (NK cells, macrophages, DCs, ?some B-cells)

SAP family of adaptors

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 frequencyof 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.

CRACC

V

C2

CD84SLAM 2B4 NTB-A

Y

V

C2

Y

Y

Y

Y

V

Y

Y

Y

V

C2

Y

Ly-9

V

C2

YY

C2

V

YY

Y

YY

Y

Ligands

V

C2

Y

Y

CD84SLAMmeasles

CD48 NTB-A Ly-9 CRACC

Y

C2

Y : TIYxxV/I motif

Y : TVYxxV/I motif YY

Y

Y

Y

SAP family adaptors associatewith SLAM-related receptors

Expression patterns of SLAM-related receptors

SAP

SLAMCD84

2B4

NTB-A

Ly-9

TH2functionsNK cell

cytotoxicity

NK cellcytotoxicity

?

?

XLP is likely caused by multipleSLAM-related receptor dysfunctions

Ig productionmemory B-cells

?

How does SAP regulate SLAM-related receptors?

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- secretion

by activated T-cells;- defective TCR-induced IL-4 production in

slam-/- mice.

SAP is required for SLAM-induced protein tyrosine phosphorylation

SH2

SH2

Lck

Zap-70

CD4 TCR

P

PFynTPP

PP P P P

PP

MHC

SH2

kinase

PP

SLAM

SLAM

SAP

SHIP-1DokRas-GAP

?Ras ? ?Itk?Akt

- -

PH

Itk

IL-4 production

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.

SH

2

SLAM

SAP

B + SAP

SH

2P

P

SAP

ligand

+ ligand

tyrosine phosphorylation

SH2

SLAM

SHP-2

A - SAP

SH2

SHP-2

ligand

+ ligand

tyrosine phosphorylation

SH2

SH2

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

Recruitment of FynT during SLAM-SAP signalling

SH2

SH2

Lck

Zap-70

CD4 TCR

P

PFynTPP

PP P P P

PP

MHC

SH2

FynT

PP

SLAM

SLAM

SAP PH

Itk

SH3SH2

How does SAP promote recruitment of FynT?

pY

P

SH3

FynT

SAP

SH2

SLAM

SH3

PP

P

SAP is a bifunctional adaptor that links SLAM to FynT

1

2

A B C D E

F G

**** ***

SH2 domain

tail

The FynT SH3 domain binds a novel arginine-basedmotif at the surface of the SAP 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

Chan et al., 2003

Crystal structure SAP SH2-FynT SH3 complex

Arginine 78 is required for SAP-mediated signalling

FynT

SLAM

SLAM

SAP

SH2Y

YYP

P

SH3

P

FynT

SAP

SH2 SH3

P

2B4

CD48

YP

YP

YP

YP

Modulation of cytotoxicityModulation of IFN-

Modulation of IL-4

SHIP-1Dok-1/2

Ras-GAP

Vav-1SHIP-1c-Cbl

A common mechanism for signalling by SLAM-related receptors

Role of FynT in SAP-dependent signalling in vivo?

fyn-/- T-cells exhibit TH2 and, to a lesser extent, TH1 cytokine defects in response to TCR stimulation

exon 1 exon 2 exon 3 exon 4

ATG TGA

SH2 domain

cgggcg

loxP

floxed sapR78A allele

Creation of a sapR78A “knock-in” mouse

sapR78A

R78A

**

IFN-

0

500

1000

1500

2000

2500

sap+

sapR78Asap-

IL-4

0

50

100

150

200

250

300

350

400

IL-13

0

50

100

150

200

250

300 Proliferation

0

50000

100000

150000

200000

250000

300000

Anti-CD3:Anti-CD28:

00

0.30

3.00

0.31

3.01

P+I Anti-CD3:Anti-CD28:

00

0.30

3.00

0.31

3.01

P+I

Anti-CD3:Anti-CD28:

00

0.30

3.00

0.31

3.01

P+IAnti-CD3:Anti-CD28:

00

0.30

3.00

0.31

3.01

P+I

IL-4

(p

g.m

l-1)

IFN

- (

pg

.ml-1

)IL

-13

(p

g.m

l-1)

cp

m

sap+

sapR78Asap-

Cytokine production defect in T-cells from sapR78A mice

0

2

4

6

8

10

0 14 28 35 42 49 70

time (days)

IgE

(g

.ml-1

) sap+

sap-

sapR78A

0123456789

IgE

(g

.ml-1

)

day 0day 14

sap+ sap- sapR78A

05

10152025303540

IgE

(g

.ml-1

)

sap- fyn-/-sapR78Acontrol

0

5

10

15

20

25

30

35

IgG

1 (

g.m

l-1)

sap- fyn-/-sapR78Acontrol

A B

C D

day 0day 14

Defective IgE production in sapR78A mice

Regulation of TH2 cytokine production by SAP-FynT pathway

DC

T-cell

MHC ?

TCR

SAP

FynT FynT

?

IL-4IL-13

Induction of SLAM expression during T-cell activation

Defective TH2 cytokine production inslam-/- T-cells

DC

T-cell

MHC SLAM

TCR

SAPFynT FynT

MHC SLAM

SLAMTCR

SAP

FynT FynT

IFN- IL-4IL-13

P

GATA-3

Regulation of TH2 cytokine production by the SLAM-SAP-FynT pathway

Conclusions1) SAP, a small SH2 domain-containing adaptor

mutated in XLP, is necessary for modulatorysignalling 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 FynTto SLAM receptors are likely to underlie at least part of the pathophysiology of XLP.

Sylvain Latour

Riyan ChenRomain RoncagalliOliver UttingJames TaylorLaurent DoucetMing-Chao ZhongMario-Ernesto Cruz-MunozMarceline Côté

Xiaochu ShiShaohua Zhang

Dominique Davidson

Collaborators:Pam SchwartzbergRusung TanYusuke YanagiLuo Yin