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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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Modulatory signalling in immune cells slam family receptors and sap related adaptors

Modulatory signalling in immune cells:

SLAM family receptors and

SAP-related adaptors

André Veillette

414A

November 2004


Modulatory signalling in immune cells slam family receptors and sap related adaptors

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


Modulatory signalling in immune cells slam family receptors and sap related adaptors

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


Modulatory signalling in immune cells slam family receptors and sap related adaptors

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


Modulatory signalling in immune cells slam family receptors and sap related adaptors

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


Modulatory signalling in immune cells slam family receptors and sap related 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 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.


  • Modulatory signalling in immune cells slam family receptors and sap related adaptors

    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



    Modulatory signalling in immune cells slam family receptors and sap related adaptors

    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



    Modulatory signalling in immune cells slam family receptors and sap related adaptors

    • 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.


  • Modulatory signalling in immune cells slam family receptors and sap related adaptors

    SAP is required for SLAM-induced

    protein tyrosine phosphorylation


    Modulatory signalling in immune cells slam family receptors and sap related adaptors

    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


    Modulatory signalling in immune cells slam family receptors and sap related adaptors

    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.


    Modulatory signalling in immune cells slam family receptors and sap related adaptors

    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?



    Modulatory signalling in immune cells slam family receptors and sap related adaptors

    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


    Modulatory signalling in immune cells slam family receptors and sap related adaptors

    SAP is a bifunctional adaptor that

    links SLAM to FynT

    SAP

    SLAM

    FynT

    SH2

    pY

    SH3

    SH3

    P

    P

    P

    P


    Modulatory signalling in immune cells slam family receptors and sap related adaptors

    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




    Modulatory signalling in immune cells slam family receptors and sap related adaptors

    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


    Modulatory signalling in immune cells slam family receptors and sap related adaptors

    Role of FynT

    in SAP-dependent signalling in vivo?


    Modulatory signalling in immune cells slam family receptors and sap related adaptors

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

    TH1 cytokine defects in response to TCR stimulation


    Modulatory signalling in immune cells slam family receptors and sap related adaptors

    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


    Modulatory signalling in immune cells slam family receptors and sap related adaptors

    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


    Modulatory signalling in immune cells slam family receptors and sap related adaptors

    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-/-


    Modulatory signalling in immune cells slam family receptors and sap related adaptors

    DC

    ?

    MHC

    TCR

    ?

    T-cell

    SAP

    FynT

    FynT

    IL-4

    IL-13

    Regulation of TH2 cytokine production by SAP-FynT pathway



    Modulatory signalling in immune cells slam family receptors and sap related adaptors

    Defective TH2 cytokine production in

    slam-/- T-cells


    Modulatory signalling in immune cells slam family receptors and sap related adaptors

    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


    Modulatory signalling in immune cells slam family receptors and sap related adaptors

    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.


    Modulatory signalling in immune cells slam family receptors and sap related adaptors

    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