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0 50 100 150 200 0. 0 0. 2 0. 4 0. 6 0. 8 1. 0 p=0.469 155 155 54 55 CD8hi HLA-Ehi CD8hi HLA-Elo CD8lo HLA-Ehi CD8lo HLA-Elo 0 50 100 150 200 0. 0 0. 2 0. 4 0. 6 0. 8 1. 0 CD8hi KLRC1hi CD8hi KLRC1lo CD8lo KLRC1hi CD8lo KLRC1lo 162 164 47 46 p=0.554 0 50 100 150 200 0. 0 0. 2 0. 4 0. 6 0. 8 1. 0 149 149 60 61 p=0.519 NCR1hi HLA-Ehi NCR1hi HLA-Elo NCR1lo HLA-Ehi NCR1lo HLA-Elo 0 50 100 150 200 0. 0 0. 2 0. 4 0. 6 0. 8 1. 0 153 155 56 55 NCR1hi KLRC1hi NCR1hi KLRC1lo NCR1lo KLRC1hi NCR1lo KLRC1lo p=0.502 Overall survival

Survival time (months)

(2)
(3)

K.

NK cells (C877)

NKG2A CD8α CD8β CD56 NKp44 NKp46 CD16 CD161 CD45RA CD45RO KLRG1 CCR7 CD127 CD27 CD28 CD103 HLA-DR CD38 CD25 PD-1 CD7 CD122 tSNE1 tSNE2 high low

J.

NK cells (C876)

NKG2A CD8α CD8β CD56 NKp44 NKp46 CD16 CD161 CD45RA CD45RO KLRG1 CCR7 CD127 CD27 CD28 CD103 HLA-DR CD38 CD25 PD-1 CD7 CD122 tSNE1 tSNE2 high low

L.

NK cells (C879)

NKG2A CD8α CD8β CD56 NKp44 NKp46 CD16 CD161 CD45RA CD45RO KLRG1 CCR7 CD127 CD27 CD28 CD103 HLA-DR CD38 CD25 PD-1 CD7 CD122 tSNE1 tSNE2 high low

H.

NK cells (C963)

NKG2A CD8α CD8β CD56 NKp44 NKp46 CD16 CD161 CD45RA CD45RO KLRG1 CCR7 CD127 CD27 CD28 CD103 HLA-DR CD38 CD25 PD-1 CD7 CD122 tSNE1 tSNE2 high low

G.

NK cells (C890)

NKG2A CD8α CD8β CD56 NKp44 NKp46 CD16 CD161 CD45RA CD45RO KLRG1 CCR7 CD127 CD27 CD28 CD103 HLA-DR CD38 CD25 PD-1 CD7 CD122 tSNE1 tSNE2 high low

NK cells (C1016)

CD8α CD8β CD56 NKp44 NKp46 CD16 CD161 CD45RA CD45RO KLRG1 CCR7 CD127 CD27 CD28 CD103 HLA-DR CD38 CD25 PD-1 CD7 CD122 tSNE1 tSNE2 low

F.

NK cells

N K G 2A N K p4 4 C D 10 3 N K p4 6 C D 7 C D 12 2 C D 16 1 C D 56 C R TH 2 TC R γ δ P D -1 c-K IT C D 28 C D 38 C D 45 R O C D 27 C D 8α C D 45 C C R 7 C D 3 C D 45 R A C D 25 C D 8β C D 16 K LG R 1 C C R 6 C D 12 7 H LA -D R -1 0 1 2

Expression ratio of NKG2A

(4)

Supplementary Figure 3.

0 8 4 * 0 4 2 * 0 4 2 * * * # (x10 4) per million * *

untreated (d24)vaccinated (d24)vaccinated (d29)vaccinated (d36) untreated (d24)vaccinated (d24)vaccinated (d29) vaccinated (d36) untreated (d24)vaccinated (d24) vaccinated (d29)vaccinated (d36)

A.

0 2 0 4 0 6 0 0 5 0 1 00 Untreated Vaccinated + NK depletion Vaccinated n.s. NK cell depletion % survival

C.

Days after tumor challenge

FSC-A SSC-A FSC-A FSC-W FSC-A Viability FSC-A CD45 NK1.1 CD3 CD8 CD4 NKG2A CD94 TIM-3 PD-1

D.

E.

0 2 0 4 0 6 0 0 1000

Days after tumor challenge

tumor size (mm

3)

NK cell depletion

CD4 T cells CD8 T cells NK cells Spleen

(5)

A.

0 2 0 4 0 6 0 0 1 00 0 Untreated Vaccinated + isotype Vaccinated + α-PD-1 tumor size (mm 3)

Days after tumor challenge

α-PD-1

B.

0 2 0 4 0 6 0 8 0 0 1 00 0 Untreated Vaccinated + isotype Vaccinated + α-NKG2A tumor size (mm 3)

Days after tumor challenge

α-NKG2A

D.

0 5 0 1 00 % NKG2A + d24 d36 d24 d36 vaccinated + isotype vaccinated + α-NKG2A CD8 T cells 0 5 0 1 00 % NKG2A + d24 d36 d24 d36 vaccinated + isotype vaccinated + α-NKG2A NK cells 0 5 0 1 00 0 5 0 1 00 0 5 0 1 00 NK cells

F.

C.

NKG2A CD94 NKG2A CD94 TIL TIL PBL CD8 T cells NK cells isotype α-NKG2A 0 2×1 04 4×1 04 d24 d36 d24 d36 vaccinated + isotype vaccinated + α-NKG2A untreated d24 CD4 TILs 0 2×1 04 4×1 04 d24 d36 d24 d36 vaccinated + isotype vaccinated + α-NKG2A untreated d24 NK TILs

E

# cells / live cells

(6)

Supplementary Figure 5.

A.

0 1 0 2 0 3 0 4 0 0 1000 tumor size (mm 3)

Days after tumor challenge

0 1 0 2 0 3 0 4 0

0 1 00

Days after tumor challenge

(7)

0 2 0 4 0 6 0 8 0 0 1000 (0/ 7) (5/ 16) (0/ 15)

Days after tumor challenge

tumor size (mm 3) TC-1, Qa-1b-/- host Untreated Vaccinated + isotype Vaccinated + α-NKG2A

A.

B.

C.

D.

E.

- CD3-CD3+ 0 4 ,00 0 8 ,00 0 * ** * splenocytes sup (25%) control 0 5 0 1 0 2 5 0 1 0 2 0 1 ,00 0 2 ,00 0 µg/ml * * * IFN-γ R

blocking Abcontrol Abisotype

0 12,500 25,000

sups unstimulatedfibroblastssplenocytesmacrophagestumor cells

gMFI * * 0 12,500 25,000

B16 RMA TC-1

Db -IFN-γ 30 U/ml IFN-γ 150 U/ml

Qa-1b Qa-1b Qa-1b

(8)

0 -103 103 104 105 APC-A :: Qa1 0 20 40 60 80 100

Normalized To Mode TC1 before

TC1Qa1 crispr 2x sort 0 -103 103 104 105 APC-A :: Qa1 0 20 40 60 80 100 Normalized To Mode 0 -103 103 104 105 APC-A :: Qa1 0 20 40 60 80 100

Normalized To Mode TC1 before

TC1Qa1 crispr 2x sort 0 -103 103 104 105 APC-A :: Qa1 0 20 40 60 80 100 Normalized To Mode TC-1 0 -103 103 104 105 APC-A :: Qa1 0 20 40 60 80 100 Normalized To Mode

Sample Name Subset Name Count 45339.fcs Single Cells 30259 45337.fcs Single Cells 30201 RMA before RMA.Qa1 Crispr 3x sort 0 -103 103 104 105 APC-A :: Qa1 0 20 40 60 80 100 Normalized To Mode

Sample Name Subset Name Count 45351.fcs Single Cells 29540 45349.fcs Single Cells 29594 0 -103 103 104 105 APC-A :: Qa1 0 20 40 60 80 100 Normalized To Mode

Sample Name Subset Name Count 45339.fcs Single Cells 30259 45337.fcs Single Cells 30201 RMA before RMA.Qa1 Crispr 3x sort 0 -103 103 104 105 APC-A :: Qa1 0 20 40 60 80 100 Normalized To Mode

Sample Name Subset Name Count 45351.fcs Single Cells 29540 45349.fcs Single Cells 29594 RMA 0 -103 103 104 105 APC-A :: Qa1 0 20 40 60 80 100 Normalized To Mode B16 0 -103 103 104 105 APC-A :: Qa1 0 20 40 60 80 100 Normalized To Mode B16Qa1Crispr 2x sort 0 -103 103 104 105 APC-A :: Qa1 0 20 40 60 80 100 Normalized To Mode B16 0 -103 103 104 105 APC-A :: Qa1 0 20 40 60 80 100 Normalized To Mode B16Qa1Crispr 2x sort B16 control staining untreated stimulated stimulated untreated Qa-1b staining Qa-1b Relative counts Before CRISPR/Cas9 After CRISPR/Cas9

A.

Supplementary Figure 7.

D.

0 .01 0 .1 1 1 0 1 00 peptide - + - + % IFN-γ / CD8 Gp100 TCR WT Qa-1b

-H.

0 5 0 1 00 C R PR NR 0 0 1 4 1 9 PFS (days): WT

untreated vaccinatedQa-1

Referenties

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