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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. 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

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  1. Modulatory signalling in immune cells: SLAM family receptors and SAP-related adaptors André Veillette 414A November 2004

  2. 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

  3. 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

  4. 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

  5. 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

  6. 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.

  7. 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

  8. Expression patterns of SLAM-related receptors

  9. 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

  10. How does SAP regulate SLAM-related receptors?

  11. 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.

  12. SAP is required for SLAM-induced protein tyrosine phosphorylation

  13. 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

  14. 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.

  15. 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?

  16. Recruitment of FynT during SLAM-SAP signalling

  17. 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

  18. SAP is a bifunctional adaptor that links SLAM to FynT SAP SLAM FynT SH2 pY SH3 SH3 P P P P

  19. 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

  20. Crystal structure SAP SH2-FynT SH3 complex Chan et al., 2003

  21. Arginine 78 is required for SAP-mediated signalling

  22. 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

  23. Role of FynT in SAP-dependent signalling in vivo?

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

  25. 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

  26. 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

  27. 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-/-

  28. DC ? MHC TCR ? T-cell SAP FynT FynT IL-4 IL-13 Regulation of TH2 cytokine production by SAP-FynT pathway

  29. Induction of SLAM expression during T-cell activation

  30. Defective TH2 cytokine production in slam-/- T-cells

  31. 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

  32. 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.

  33. 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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