CIS INTERACTION IN NK CELLS

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

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

3. Activating Inhibitory adhesion INTRODUCTION: NK cell Immunological Synapse (NKIS) 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. S total = S+ - Inhibited (S+)

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

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

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

7. INTRODUCTION: NK cell Immunological Synapse (NKIS) 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.

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

9. 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 Cis interaction 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 (2M) (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 Cis interaction

11. Cis interaction 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 Cis interaction

13. 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. Cis interaction: Goals and Methods

14. 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). Cis interaction: Goals and Methods. 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. Steady-State Integrated Signals ▲ - 0 MICA, a ■ – 1000MICA, a ♦ – 3000 MICA, and a ● – 5000 MICA. parameters investigation : MHC

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

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

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

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

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

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

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

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

25. 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). conclusions :

26. presented by Refael Cohen under supervision of: Prof. Ramit Mehr Asya Kaplan