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

CIS INTERACTION IN NK CELLS

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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. INTRODUCTION: NK cell Immunological Synapse (NKIS). The receptors on NK cell: - PowerPoint PPT Presentation

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Page 1: CIS INTERACTION IN NK CELLS

CIS INTERACTION IN NK CELLS

Presented by Refael CohenPresented by Refael CohenProject Advisor: Prof. Ramit Mehr

Asya Kaplan

Bar-Ilan UniversityFaculty of Life SciencesComputational Biology

2009

Presented by Refael CohenPresented by Refael CohenProject Advisor: Prof. Ramit Mehr

Asya Kaplan

Bar-Ilan UniversityFaculty of Life SciencesComputational Biology

2009

Page 2: CIS INTERACTION IN NK CELLS

The receptors on NK cell: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)

Page 3: CIS INTERACTION IN NK CELLS

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 adhesionActivating Inhibitory adhesion

Page 4: CIS INTERACTION IN NK CELLS

Active Synapse:

The order: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)

Page 5: CIS INTERACTION IN NK CELLS

Active Synapse: LFA1 binds to ICAM. The number of

bound adhesion

molecules > Cmin.

INTRODUCTION: NK cell Immunological Synapse (NKIS)

Page 6: CIS INTERACTION IN NK CELLS

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)

Page 7: CIS INTERACTION IN NK CELLS

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)

Page 8: CIS INTERACTION IN NK CELLS

Active Synapse: Switch point of NKG2D molecules.

INTRODUCTION: NK cell Immunological Synapse (NKIS)

Num

ber

of

NK

G2D

Num

ber

of

NK

G2D

Time stepTime step

• cSMACcSMAC

• pSMSCpSMSC

Page 9: CIS INTERACTION IN NK CELLS

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

Page 10: CIS INTERACTION IN NK CELLS

Cis interaction

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

Page 11: CIS INTERACTION IN NK CELLS

Cis interaction

Cis interaction

Page 12: CIS INTERACTION IN NK CELLS

Cis interaction

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

Page 13: CIS INTERACTION IN NK CELLS

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.

Page 14: CIS INTERACTION IN NK CELLS

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. MOVIEMOVIE

Page 15: CIS INTERACTION IN NK CELLS

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.

Page 16: CIS INTERACTION IN NK CELLS

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

0 2000 4000 6000-500

0

500

1000

1500

2000

2500

30002000KIR2000NKG2D

0 2000 4000 6000-500

0

500

1000

1500

2000

2500

3000

2000KIR1000NKG2D

0 2000 4000 6000-500

0

500

1000

1500

2000

2500

30001000KIR2000NKG2D

0 2000 4000 6000-500

0

500

1000

1500

2000

2500

3000

1000KIR1000NKG2D

0 2000 4000 6000-500

0

500

1000

1500

2000

2500

3000

2000KIR2000NKG2D

0 2000 4000 6000-500

0

500

1000

1500

2000

2500

30002000KIR1000NKG2D

0 2000 4000 6000-500

0

500

1000

1500

2000

2500

30001000KIR2000NKG2D

0 2000 4000 6000-500

0

500

1000

1500

2000

2500

3000

1000KIR1000NKG2D

0 2000 4000 6000-500

0

500

1000

1500

2000

2500

30002000KIR2000NKG2D

0 2000 4000 6000-500

0

500

1000

1500

2000

2500

30002000KIR1000NKG2D

0 2000 4000 6000-500

0

500

1000

1500

2000

2500

3000

1000KIR2000NKG2D

0 2000 4000 6000-500

0

500

1000

1500

2000

2500

30001000KIR1000NKG2D

0 2000 4000 6000-500

0

500

1000

1500

2000

2500

30002000KIR2000NKG2D

0 2000 4000 6000-500

0

500

1000

1500

2000

2500

3000

2000KIR1000NKG2D

0 2000 4000 6000-500

0

500

1000

1500

2000

2500

30001000KIR2000NKG2D

0 2000 4000 6000-500

0

500

1000

1500

2000

2500

3000

1000KIR1000NKG2D

0 2000 4000 6000-500

0

500

1000

1500

2000

2500

30002000KIR2000NKG2D

0 2000 4000 6000-500

0

500

1000

1500

2000

2500

30002000KIR1000NKG2D

0 2000 4000 6000-500

0

500

1000

1500

2000

2500

30001000KIR2000NKG2D

0 2000 4000 6000-500

0

500

1000

1500

2000

2500

30001000KIR1000NKG2D

A. R

adiu

s 1

B. R

adiu

s 3

C

. Rad

ius

5

D. R

adiu

s 10

E

. Rad

ius

50

MHCMHC

Ste

ady-

Sta

te I

nte

gra

ted

Sig

nal

s

Page 17: CIS INTERACTION IN NK CELLS

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

0 2000 4000 6000-500

0

500

1000

1500

2000

2500

30001000KIR2000NKG2D

MHCMHC

Inte

grat

ed s

igna

lIn

tegr

ated

sig

nal

Page 18: CIS INTERACTION IN NK CELLS

0 2000 4000 6000-500

0

500

1000

1500

2000

2500

30002000KIR2000NKG2D

0 2000 4000 6000-500

0

500

1000

1500

2000

2500

3000

2000KIR1000NKG2D

0 2000 4000 6000-500

0

500

1000

1500

2000

2500

30001000KIR2000NKG2D

0 2000 4000 6000-500

0

500

1000

1500

2000

2500

3000

1000KIR1000NKG2D

0 2000 4000 6000-500

0

500

1000

1500

2000

2500

3000

2000KIR2000NKG2D

0 2000 4000 6000-500

0

500

1000

1500

2000

2500

30002000KIR1000NKG2D

0 2000 4000 6000-500

0

500

1000

1500

2000

2500

30001000KIR2000NKG2D

0 2000 4000 6000-500

0

500

1000

1500

2000

2500

3000

1000KIR1000NKG2D

0 2000 4000 6000-500

0

500

1000

1500

2000

2500

30002000KIR2000NKG2D

0 2000 4000 6000-500

0

500

1000

1500

2000

2500

30002000KIR1000NKG2D

0 2000 4000 6000-500

0

500

1000

1500

2000

2500

3000

1000KIR2000NKG2D

0 2000 4000 6000-500

0

500

1000

1500

2000

2500

30001000KIR1000NKG2D

0 2000 4000 6000-500

0

500

1000

1500

2000

2500

30002000KIR2000NKG2D

0 2000 4000 6000-500

0

500

1000

1500

2000

2500

3000

2000KIR1000NKG2D

0 2000 4000 6000-500

0

500

1000

1500

2000

2500

30001000KIR2000NKG2D

0 2000 4000 6000-500

0

500

1000

1500

2000

2500

3000

1000KIR1000NKG2D

0 2000 4000 6000-500

0

500

1000

1500

2000

2500

30002000KIR2000NKG2D

0 2000 4000 6000-500

0

500

1000

1500

2000

2500

30002000KIR1000NKG2D

0 2000 4000 6000-500

0

500

1000

1500

2000

2500

30001000KIR2000NKG2D

0 2000 4000 6000-500

0

500

1000

1500

2000

2500

30001000KIR1000NKG2D

A. R

adiu

s 1

B. R

adiu

s 3

C

. Rad

ius

5

D. R

adiu

s 10

E

. Rad

ius

50

MHCMHC

▲ - 0 MICA, a ■ – 1000MICA, a ♦ – 3000 MICA, and a ● – 5000 MICA. parameters investigation: S

tead

y-S

tate

In

teg

rate

d S

ign

als

Page 19: CIS INTERACTION IN NK CELLS

parameters investigation : 1000 MHC on NK cell and target cell (30% cis)1000 MHC on NK cell and target cell (30% cis)::

Activating Integrated signal: x2Activating Integrated signal: x2Ly49-MHC trans

0

1020

30

4050

60

70

8090

100

0 5000 10000 15000 20000 25000 30000 35000

Without cis:

Ly49-MHC trans

010

20

30

40

50

60

7080

90

100

0 5000 10000 15000 20000 25000 30000 35000

With cis: integrated signal

0

50

100

150

200

250

300

0 5000 10000 15000 20000 25000 30000 35000

With cis:

integrated signal

0

50

100

150

200

250

300

0 5000 10000 15000 20000 25000 30000 35000

Without cis:

pSMAC pSMAC cSMAC cSMAC

Page 20: CIS INTERACTION IN NK CELLS

Number of NKG2D and MICA: a bit more in centerNumber of NKG2D and MICA: a bit more in centerMICA

SP=1200

0

100

200

300

400

500

600

700

800

0 5000 10000 15000 20000 25000 30000 35000

Without cis:

NKG2DSP=2300

0

100

200

300

400

500

600

700

800

0 5000 10000 15000 20000 25000 30000 35000

With cis: MICASP=1000

0

100

200

300

400

500

600

700

800

0 5000 10000 15000 20000 25000 30000 35000

With cis:

NKG2DSP=2300

0

100

200

300

400

500

600

700

800

0 5000 10000 15000 20000 25000 30000 35000

Without cis:

parameters investigation : 1000 MHC on NK cell and target cell (30% cis)1000 MHC on NK cell and target cell (30% cis)::

pSMAC pSMAC cSMAC cSMAC

Page 21: CIS INTERACTION IN NK CELLS

Number of Ly49 and MHC: a bit less in centerNumber of Ly49 and MHC: a bit less in centerLy49

0

100

200

300

400

500

600

700

800

0 5000 10000 15000 20000 25000 30000 35000

Without cis: MHC on target

0

100

200

300

400

500

600

700

800

0 5000 10000 15000 20000 25000 30000 35000

Without cis:

Ly49

0

100

200

300

400

500

600

700

800

0 5000 10000 15000 20000 25000 30000 35000

With cis: MHC on target

0

100

200

300

400

500

600

700

800

0 5000 10000 15000 20000 25000 30000 35000

With cis:

parameters investigation : 1000 MHC on NK cell and target cell (30% cis)1000 MHC on NK cell and target cell (30% cis)::

pSMAC pSMAC cSMAC cSMAC

Page 22: CIS INTERACTION IN NK CELLS

Number of LFA-1 and ICAM: later switchNumber of LFA-1 and ICAM: later switch

LFA-1SP=2800

050

100150200250300350400450

0 5000 10000 15000 20000 25000 30000 35000

Without cis: ICAM

0

50

100

150

200

250

300

350

400

0 5000 10000 15000 20000 25000 30000 35000

Without cis:

LFA-1SP=3200

0

50

100

150

200

250

300

350

400

450

0 5000 10000 15000 20000 25000 30000 35000

With cis: ICAM

0

50

100

150

200

250

300

350

400

0 5000 10000 15000 20000 25000 30000 35000

With cis:

parameters investigation : 1000 MHC on NK cell and target cell (30% cis)1000 MHC on NK cell and target cell (30% cis)::

pSMAC pSMAC cSMAC cSMAC

Page 23: CIS INTERACTION IN NK CELLS

parameters investigation :

Effects of varying the directed movement factors on Effects of varying the directed movement factors on molecule distributions.molecule distributions.

Without cis:Without cis:

0 2 4 6 8 10 120

20

40

60

80

100

0 2 4 6 8 10 120

20

40

60

80

100

0 2 4 6 8 10 120

20

40

60

80

100

0 2 4 6 8 10 120

20

40

60

80

100

0 2 4 6 8 10 120

20

40

60

80

100

0 2 4 6 8 10 120

20

40

60

80

100

120

0 2 4 6 8 10 120

20

40

60

80

100

120

0 2 4 6 8 10 120

20

40

60

80

100

120

0 2 4 6 8 10 120

20

40

60

80

100

0 2 4 6 8 10 120

20

40

60

80

100

0 2 4 6 8 10 125

10

15

20

25

30

35

0 2 4 6 8 10 125

10

15

20

25

30

35

0 2 4 6 8 10 125

10

15

20

25

30

35

0 2 4 6 8 10 120

10

20

30

40

0 2 4 6 8 10 120

10

20

30

40

1 1.5 2 5 10 .

% N

KG

2D in

ce

nte

r

% K

IR in

ce

nte

r

% L

FA

-1 in

ce

nte

r

Activated receptor and ligand directed movement factor:

Raft: ▲: 10; ■: 5; ♦: 2; ●: 1Raft: ▲: 10; ■: 5; ♦: 2; ●: 1

0 2 4 6 8 10 120

20

40

60

80

100

120

0 2 4 6 8 10 120

20

40

60

80

100

120

Inhibition receptor and ligandInhibition receptor and ligand

Page 24: CIS INTERACTION IN NK CELLS

parameters investigation:

Effects of varying the directed movement factors on Effects of varying the directed movement factors on molecule distributions.molecule distributions.

With cis:With cis:

0 2 4 6 8 1 0 1 20

2 0

4 0

6 0

8 0

1 0 0

I n h ib it o r y D M0 2 4 6 8 1 0 1 2

0

2 0

4 0

6 0

8 0

1 0 0

0 2 4 6 8 1 0 1 20

2 0

4 0

6 0

8 0

1 0 0

0 2 4 6 8 1 0 1 20

2 0

4 0

6 0

8 0

1 0 0

0 2 4 6 8 1 0 1 20

2 0

4 0

6 0

8 0

1 0 0

1 2 0

0 2 4 6 8 1 0 1 20

2 0

4 0

6 0

8 0

1 0 0

1 2 0

0 2 4 6 8 1 0 1 20

2 0

4 0

6 0

8 0

1 0 0

1 2 0

0 2 4 6 8 1 0 1 20

2 0

4 0

6 0

8 0

1 0 0

1 2 0

0 2 4 6 8 1 0 1 20

2 0

4 0

6 0

8 0

1 0 0

0 2 4 6 8 1 0 1 20

2 0

4 0

6 0

8 0

1 0 0

0 2 4 6 8 1 0 1 25

1 0

1 5

2 0

2 5

3 0

3 5

0 2 4 6 8 1 0 1 25

1 0

1 5

2 0

2 5

3 0

3 5

0 2 4 6 8 1 0 1 25

1 0

1 5

2 0

2 5

3 0

3 5

0 2 4 6 8 1 0 1 25

1 0

1 5

2 0

2 5

3 0

3 5

0 2 4 6 8 1 0 1 20

1 0

2 0

3 0

4 0

Activated receptor and ligand directed movement factor:

% N

KG

2D

in c

ente

r

% K

IR in

ce

nter

% L

FA-1

in

cen

ter

11 1.5 2 5 10 1.5 2 5 10

. .

Activated receptor and ligand directed movement factor:

Raft: ▲: 10; ■: 5; ♦: 2; ●: 1Raft: ▲: 10; ■: 5; ♦: 2; ●: 1

0 2 4 6 8 10 120

20

40

60

80

100

120

0 2 4 6 8 10 120

20

40

60

80

100

120

Inhibition receptor and ligandInhibition receptor and ligand

Page 25: CIS INTERACTION IN NK CELLS

conclusions:

Conclusions:Conclusions:

Very increasing in integrated signal in program with cis Very increasing in integrated signal in program with cis interaction.interaction.

The synapse not faster with cis (switch point of NKG2D The synapse not faster with cis (switch point of NKG2D not faster) because integrated signal not very increase not faster) because integrated signal not very increase in initial of the synapse.in initial of the synapse.

Less Ly49 in cSMAC with cis, maybe because Less Ly49 in cSMAC with cis, maybe because interruption of cis complexes to Ly49 to move to center interruption of cis complexes to Ly49 to move to center (no Directed Movement to Ly49).(no Directed Movement to Ly49).

As less Ly49 in cSMAC as more NKG2D in cSMAC – less As less Ly49 in cSMAC as more NKG2D in cSMAC – less crowded in center.crowded in center.

Switch point of LFA1 is slower with cis, maybe because Switch point of LFA1 is slower with cis, maybe because interruption of cis complexes to LFA1 to move to center interruption of cis complexes to LFA1 to move to center (no Directed Movement to LFA1).(no Directed Movement to LFA1).

Page 26: CIS INTERACTION IN NK CELLS