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rFibronectin-FcrE-cadh-FcrICAM-1-Fc

rICAM-1-Fc rE-cadh-Fc rFibronectin-Fc

-

+ rTGF-β

96 h 24 h

30 min

rE-cadh-FcrICAM-1-Fc

+ rCXCL12

rFibronectin-Fc

-

-

rICAM-1-Fc rE-cadh-Fc

6 h 0

75

Mean CTL number per field

150 225 300 375

* *

**

**

+ rTGF-β

* *

Mean CTL shape index 0 0.8 1.6 2.4 3.2

4 *

*

**

***

*

***

* *

*

**

rE-cadh-Fc

+ rTGF-β

rICAM-1-Fc

-

MeanT-cell number per field

0 50 100 150 200

250 -

+ rTGF-β

B

+ rTGF-β(6 h) + rTGF-β(96 h)

+ rTGF-β(30 min) + rTGF-β(24 h)

T T T

T S

S S

IGR-Heu S

CTL CD103^-/LFA-1⁺

Stroma Stroma

T S

IGR-Heu-ICAM-1

- + anti-β₂

T S

+ anti-CD3 + anti-CD103

CTL CD103^-/LFA-1⁺

+ rTGF-β(96 h)

T S T

S

+ rTGF-β(30 min) + rTGF-β(6 h) + rTGF-β(24 h)

B

T T T

T S

S S

S

S T

IGR-Heu

CTL CD103⁺/LFA-1⁺

Stroma

T

+ anti-CD103

S

S T

+ anti-β₂ + anti-CD103 + anti-β2

S T T

S

IGR-Heu-ICAM-1

T

S S T

S T T

S

-

-

% CTL in tumor islets

0 25 50 75

**

****

% CTL in tumor islets

0 25 50 75

****

* **

0 25 50

75 ** *

**

**

***

+ rTGF-β CD103⁺/LFA-1⁺

% CTL in tumor islets

CTL CD103^-

/LFA-1⁺

B

***

Ca²+ratio

***

1.5 2.5 3.5 4.5

CTL

***

CD103⁺/LFA-1⁺ CD103^-/LFA-1⁺

IGR-Heu-ICAM-1

CTL - - ^{+ anti-E2 + rTGF-E}

-

+ anti-CD103

+ anti-E2 + rTGF-E

4.5 Color merge Ca2+ratio

***

CTL Ca²+ratio

******

1.5 2.5 3.5

4.5 *** **

CD103⁺/LFA-1⁺ CD103^-/LFA-1⁺ CD103-/LFA-1+ CD103+/LFA-1+ + CD103-/LFA-1+

Color merge Ca2+ratio

4.5

0 CD103+/LFA-1+ CD103-/LFA-1+ CD103+/LFA-1+ + CD103-/LFA- 1+ CD103-/LFA- 1+

- -

- - CD103^-/LFA-1⁺

CD103⁺/LFA-1⁺ CD103^-/LFA-1⁺

CD103+/LFA-1+ CD103-/LFA-1+

B

+ rEcadh-Fc Medium

0

phospho-ILK/ILK ratio

0.5 1 1.5

ILK

Phospho-ILK 54 kDa

52 kDa 42 kDa F-actin

ILK Phospho-ILK

F-actin

+ rE-cadh-Fc

+ rTGF-E

1

0 0.2 0.4 0.6 0.8

+ rE-cadh-Fc

Phospho-ILK/ILK ratios

+ rTGF-E

52 kDa 42 kDa 54 kDa

1

0 0.2 0.4 0.6 0.8

+ rTGF-β

Phospho-ILK/ILK ratios

+ rE-cadh-Fc

62 kDa 60 kDa 42 kDa Phospho-AKT

AKT F-actin

C

1

0

+ rE-cadh-fc 0.2

0.4 0.6 0.8

Phospho-AKT/AKT ratio

C D

IP: anti-CD103 mAb

52 kDa Anti-CD103

IgG WB: ILK

Input

IP: anti-TGFBRI mAb

WB: ILK 52 kDa

Input

B

+ QLTDMSO

Phospho-ILK Merge Contrast

IGR-Heu + CTL CD103⁺/LFA-1⁺ TGFBRI

CD103

+ SB % CTL with polarized proteins

**

0 40 20 60 80

100 ******

****

Phospho-ILK TGFBRI CD103

+ DMSO + QLT (20µM) + SB (1µM) % CTL with phospho-ILK relocalized at the leading edge

0 40

20 60 80

CTL CD103⁺/LFA-1⁺ CD103^-/LFA-1⁺

CD103⁺/ LFA-1⁺

CD103^- /LFA-1⁺ Anti-TGFBRI IP: anti-TGFBRI mAb

WB: anti-

CTL

E

B

C _CD103-/LFA-1⁺CTL

+ IGR-Heu-ICAM-1

Medium + rTGF-E CD103⁺/LFA-1⁺CTL

+ IGR-Heu

0 15 30 45 60

30:1 10:1 3:1 E:T ratio

* *

S T

S T S

T

IGR-Heu

+ SB + MK-2206

S

T S

T

E

0 25 50 75

****

% CTL in tumor islets

0 25 50 75 100

% CD107a+ CTL + rE-cadh-Fc + anti-CD3 mAb

+ rICAM-1-Fc + anti-CD3 mAb + anti-CD3 mAb (10µg/mL)

******

**

*

**

- + SB

Medium + Wortm

β

% specific lysis

0 15 30 60

30:1 10:1 3:1

45 *

* *

E:T ratio

CD103⁺/LFA-1⁺CTL + IGR-Heu

D

Medium +rTGF-β

+rTGF-β + anti-CD103

30:1 10:1 3:1

0 15 30 60

45

**

**

E:T ratio