0.23

0.35

TIGIT-APC

LAG

-3-B

V42

1

TIGIT-APC

TIM

-3-P

E

Supplemental Figure 1

3.36

0.93

Spleen(WT) Spleen(Con) TIL(Con)

Gated on CD8+A

B

TIGIT-APC

LAG

-3-B

V42

1

TIGIT-APC

TIM

-3-P

E

4.57

Spleen(WT) Spleen(Con) TIL(Con)

Gated on CD4+

2.34

0.99

6.63

0.80 4.09

1.89 2.53

Supplemental Figure 2

B

Pre-sorting

Post-sorting

CD3-FITC

CD

8-P

erC

P-C

y5.5

8.4877.5799.1457.46

A

Pre-sorting

Post-sorting

FSC-A

SS

C-A

CD4-FITC

SS

C-A

FSC-A

FS

C-W

93.70 96.90 26.70 14.88

CD25-PE

Fox

p3-

Per

CP

-Cy5

.5

FSC-H

FS

C-W

99.53

FSC-A

SS

C-A

CD4-FITCS

SC

-AFSC-A

FS

C-W

93.06 96.93 93.95 95.56

CD25-PE

Fox

p3-

Per

CP

-Cy5

.5

FSC-H

FS

C-W

98.62

FSC-A

SS

C-A

FSC-A

FS

C-W

FSC-H

FS

C-W

CD3-FITC

CD

8-P

erC

P-C

y5.5

FSC-A

SS

C-A

FSC-A

FS

C-W

FSC-H

FS

C-W

99.00100.0099.7181.57

Supplemental Figure 3

23

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67

82

34

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*

HNSC(num(T)=519; num(N)=44)

0 50 100 150 200

0.0

0.2

0.4

0.6

0.8

1.0

Overall Survival

MonthsP

erc

en

t su

rviv

al

Low PVR TPMHigh PVR TPM

Logrank p=0.0023 HR(high)=1.5

p(HR)=0.0025 n(high)=259

n(low)=259

BA

Supplemental Figure 4

HE HE CD155 CD155

HE HE CD155 CD155

HE HE CD155 CD155 50 m

A

B Cn=35

LNHNSCC

300

200

100

0

300

200

100

0

T1 + T2 T3 + T4

P = 0.9148 n=67

n=145P = 0.1122

Supplemental Figure 5

50μm

CD155

PDL1

DAPI

CD11B

CD155-APC

SS

C-A

Supplemental Figure 6

FVD-eFlour506

SS

C-A

FSC-A

SS

C-A

FSC-A

FS

C-W

CD11b-FITC

SS

C-A

A

BCD155-APC

SS

C-A

FMO-CD155

FMO-CD155

CD155

CD155

Pre-sorting Post-sorting

91.3920.40

CD11b-FITC

SS

C-A

CD11b-FITC

SS

C-A

Ly6G-PE

Ly6C

-P

E-C

y7

7.61

78.9891.01

99.40

98.83

PMN-MDCS

M-MDSC

Ly6G-PE

Ly6C

-P

E-C

y7

Ly6G-PE

Ly6C

-P

E-C

y7

Supplemental Figure 7

A

ISO

Ant

i-TIG

IT

Liver Kidney

100μm

0

Gr-

1+(%

) on

CD

11b+

cells

B

20

40

60ISO

Anti-TIGIT

P = 0.2295

P = 0.3945

Spleen Tumor

Fox

p3-

Per

CP

-Cy5

.5

Supplemental Figure 8

3.64

6.39 0.05

0.26

14.45

9.18

27.35

1.12

0.18

0.11

CD4-FITC

SS

C-A

CD8-PerCP-Cy5.5

SS

C-A

CD11b-FITC

Gr1

-AP

C

CD25-PE

Gated on CD4+

Isotype Anti-CD4

Isotype Anti-CD8

A B

C

Isotype Anti-Gr1

Isotype Anti-CD25

Blood

LN

D

0

TIG

IT+(%

) on

CD

8+ce

lls

20

40

60

ISO

Anti-PD1

P = 0.4565

P = 0. 2700

Spleen Tumor