1. CIS INTERACTION IN NK CELLS Presented by Refael Cohen Project Advisor: Prof. Ramit Mehr Asya Kaplan Bar-Ilan University Faculty of Life Sciences Computational Biology 2009 Presented by Refael Cohen Project Advisor: Prof. Ramit Mehr Asya Kaplan Bar-Ilan University Faculty of Life Sciences Computational Biology 2009

2. The receptors on NK cell: Adhesion molecules – LFA-1/ICAM-1Adhesion molecules – LFA-1/ICAM-1 Activating complexes – NKG2D/MICAActivating complexes – NKG2D/MICA Inhibitory complexes – Ly49/MHC-1Inhibitory complexes – Ly49/MHC-1 Lipid raftsLipid rafts INTRODUCTION: NK cell Immunological Synapse (NKIS)

3. Active Synapse:  Increase in integrated activating signal. Our model: Each bound inhibitory receptor inhibits all the bound activating receptors within a Certain radius.  Lysis of target cell. INTRODUCTION: NK cell Immunological Synapse (NKIS) S total = S+ - Inhibited (S+) Activating Inhibitory adhesion

4. Active Synapse: The order: 1.LFA1 binds to ICAM. 2.The number of bound adhesion molecules > Cmin. 3.NKG2D and Ly49 bind their ligands. 4.Smin < integrated activating signal. 5.Directed movement of NKG2D & lipid rafts. 6.Surrounding ring of LFA1 – stress of membrane. 7.Switch point of NKG2D molecules. 8.Increase in integrated activating signal. 9.Lysis of target cell. INTRODUCTION: NK cell Immunological Synapse (NKIS)

5. Active Synapse:  LFA1 binds to ICAM.  The number of bound adhesion molecules > Cmin. INTRODUCTION: NK cell Immunological Synapse (NKIS)

6. Active Synapse:  NKG2D and Ly49 bind their ligands.  When integrated activating signal > Smin - Directed movement of NKG2D & lipid rafts. INTRODUCTION: NK cell Immunological Synapse (NKIS)

7. Active Synapse:  Surrounding ring of LFA1 - Short and long complexes in adjacent membrane sites may cause a structural stress in the membrane, pushing the long ones (LFA1 complex) out from the short ones. INTRODUCTION: NK cell Immunological Synapse (NKIS)

8. Active Synapse:  Switch point of NKG2D molecules. INTRODUCTION: NK cell Immunological Synapse (NKIS) Number of NKG2D Time step cSMAC cSMAC pSMSC pSMSC

9. Cis interaction  The inhibitory Ly49A NK cell receptor not only binds to its H-2Dd ligand expressed on potential target cells (in trans) but also is constitutively associated with H-2Dd in cis (on the same cell).  This binding probably does not transduce an inhibitory signal. Cis interaction

10.  The long Ly49 stalk region (67-137 aa) and 3  -helices shows considerable flexibility.  Cis association and trans interaction occur through the same binding site (  2 M) (Marie-Agnes Douceyl, Leonardo Scarpellino,, Jacques Zimmer, Philippe Guillaume, Immanuel Luescher, Claude Bron & Werner Held, Cis association of Ly49A with MHC class I restricts natural killer cell inhibitionj, Nature Immuno. 2004) Cis interaction

12.  Cis association restricts the number of Ly49 receptors available for binding of H-2Dd on target cells and reduces NK cell inhibition through Ly49.  The result: more activating integrated signal, and faster lysis. Cis interaction

13. Cis interaction: Goals and Methods My projects: Part 1: To update the simulation of NK cell synapse (over 10,000 C++ code lines) with cis interaction by hundreds of additions and changes, include graphic exhibit.

14. Cis interaction: Goals and Methods. Part 2: To run the update program and find when will accept positive synapse. Also to compare the results with old program (without cis interaction). Part 2:   To run the update program and check What are the effects of cis interaction on behavior of synapse?   Also to compare the results with old program (without cis interaction).   To draft graphs with MATLAB programs. MOVIE

15. Cis interaction: Goals and Methods.   Parameters:   cis interaction exist in mouse only, and old program was on human, therefore we need to find new data for synapse.   We find data from article on: bond length, diffusion rate, microdomain preference, dissociation rate, association rate of all percipient molecules.   Other data remain as parameters.

16. ▲ - 0 MICA, a ■ – 1000MICA, a ♦ – 3000 MICA, and a ● – 5000 MICA. parameters investigation : MHC Steady-State Integrated Signals

17. ▲ - 0 MICA, a ■ – 1000MICA, a ♦ – 3000 MICA, and a ● – 5000 MICA. parameters investigation : MHC Integrated signal

18. MHC ▲ - 0 MICA, a ■ – 1000MICA, a ♦ – 3000 MICA, and a ● – 5000 MICA. parameters investigation : Steady-State Integrated Signals

19. 1000 MHC on NK cell and target cell (30% cis): parameters investigation : 1000 MHC on NK cell and target cell (30% cis): Activating Integrated signal: x2 Without cis:With cis: Without cis: pSMAC cSMAC pSMAC cSMAC

20. Number of NKG2D and MICA: a bit more in center Without cis: With cis: Without cis: 1000 MHC on NK cell and target cell (30% cis): parameters investigation : 1000 MHC on NK cell and target cell (30% cis): pSMAC cSMAC pSMAC cSMAC

21. Number of Ly49 and MHC: a bit less in center Without cis: With cis: 1000 MHC on NK cell and target cell (30% cis): parameters investigation : 1000 MHC on NK cell and target cell (30% cis): pSMAC cSMAC pSMAC cSMAC

22. Number of LFA-1 and ICAM: later switch Without cis: With cis: 1000 MHC on NK cell and target cell (30% cis): parameters investigation : 1000 MHC on NK cell and target cell (30% cis): pSMAC cSMAC pSMAC cSMAC

23. parameters investigation : Effects of varying the directed movement factors on molecule distributions. Without cis: Activated receptor and ligand directed movement factor: Raft: ▲: 10; ■: 5; ♦: 2; ●: 1 Inhibition receptor and ligand

24. parameters investigation : Effects of varying the directed movement factors on molecule distributions. With cis: 1 1.5 2 5 10. 1 1.5 2 5 10. Activated receptor and ligand directed movement factor: Raft: ▲: 10; ■: 5; ♦: 2; ●: 1 Inhibition receptor and ligand

25. conclusions : Conclusions:  Very increasing in integrated signal in program with cis interaction.  The synapse not faster with cis (switch point of NKG2D not faster) because integrated signal not very increase in initial of the synapse.  Less Ly49 in cSMAC with cis, maybe because interruption of cis complexes to Ly49 to move to center (no Directed Movement to Ly49).  As less Ly49 in cSMAC as more NKG2D in cSMAC – less crowded in center.  Switch point of LFA1 is slower with cis, maybe because interruption of cis complexes to LFA1 to move to center (no Directed Movement to LFA1).