1

SUPPLEMENTAL MATERIAL AND METHODS

Cytokine analysis

Serum from treated animals was collected and a multiplex assay of the mouse cytokine magnetic 10-Plex

Panel (Luminex Life-Technologies©) was performer for quantitative cytokine measurement according

manufacture’s instructions.

Quantification of MCMV

At the indicated time points of infection, spleen, liver and SG were collected and DNA was extracted

using DNeasy Tissue kit following manufacture’s instructions. Quantification of MCMV titers was

performed by qPCR as previously described (1). The data represents IE1 gene copies per organ.

Microarray Analysis

RNA from freshly isolated NK cells or activated adherent NK cells was extracted using RNeasy Mini kit

(Qiagen) with on-column DNase step (Qiagen) per the manufacturer’s instructions. RNA was then

quantified using the DS-11 FX+ Spectrophotometer / Fluorometer (Denovix Inc., Wilmington, DE). RNA

(150–300 ng) was used for generating biotinylated cRNA through single-round amplification using the

MessageAmpIII RNA Amplification kit following the manufacturer’s recommendations (Ambion,

ThermoFisher Scientific Inc.). A total of 20 μg of biotinylated cRNA was fragmented and hybridized to

Affymetrix murine Mouse Gene 2.0ST (ThermoFisher Scientific Inc.) and scanned at the Protein and

Nucleic Acid Facility (Stanford University). Two independent RNA samples were analyzed. The

microarray data were analyzed using the Affymetrix Transcriptome Analysis Console. Microarray probe

intensity values (CEL files) were background-corrected, summarized and normalized using the Robust

Multi-array Average (RMA16) algorithm 32 and subsequently filtered on raw intensity for data in the

upper 20-100th percentile using the Expression Suite Software version 1.1 (ThermoFisher Scientific Inc.).

We considered genes differentially regulated between freshly isolated NK cells (day 0) and the other

conditions that were more than 1.5 fold differentially expressed with a false discovery rate equal to or less

than 0.1 and with an ANOVA significance value of equal to or smaller than 0.05. Ingenuity® Pathway

Analysis (IPA®) software (Qiagen, Redwood City, CA) was used to predict the biological processes that

2

the up-regulated genes could be involved in. PCA was performed using ggfortify and ggplot package of

Rstudio. The two largest principal components were plotted against one another to assess the relationships

between different biological repeats of each sample. A Venn diagram of only up-regulated genes was also

displayed using the VennDiagram package in Rstudio. The microarray data has been deposited in the

NCBI’s Gene Expression Omnibus database (Edgar et al., 2002) and are accessible throught GEO Series

accession number GSE131522 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE131522).

In vivo evaluation of NK cell proliferation

C57BL/6 L2G85 Luc+ fresh NK cells or ex vivo IL-2 stimulated aNK cells (control aNK or KU treated)

were transfered iv. into BALB/c Rag2-/-IL2Rc-/- deficient mice and treated with 5x104 IU IL-2 for 7 days.

NK cell proliferation, expansion and survival were measured by bioluminescence at different time points.

SUPPLEMENTAL MATERIAL AND METHODS’ REFERENCES

1. M. Alvarez et al., Contrasting effects of anti-Ly49A due to MHC class I cis binding on NK cell-mediated allogeneic bone marrow cell resistance. Journal of immunology 191, 688 (Jul 15, 2013).

Antibody Clone Provider Catalogue

Armenian Hamster IgG Thermo Fisher Scientific 13-4113-85

Armenian Hamster IgG Isotype Control

eBio299Arm Thermo Fisher Scientific 14-4888-81

ATM (pSer1981) 10H11.E12 Thermo Fisher Scientific 50-9046-41

BCL2 BCL/10C4 BioLegend 633509

CD122 5H4 BioLegend 105904

CD3 145-2C11 BioLegend 100334, 100355

CD4 RM4-5 BioLegend 100552

CD49b DX5 BioLegend 108906

CD8 53-6.7 BioLegend 100714, 100744

CD96 3.3 BioLegend 131705

Chk2 (pT68) Abcam ab85743

DNAM1 1.00E+06 BioLegend 128803

Eomes Dan11mag BioLegend 12-4875-82

FasL MFL3 Thermo Fisher Scientific 46-5911-82

Granzyme B GB11 BioLegend 515403

IFNg XMG1.2 BioLegend 505814, 505837

ISo BioLegend

Ki67 16A8 BioLegend 652420

KLRG1 2F1/KL1261 BioLegend 138409, 138423

Ly49G2 4D11 BD Bioscience 555315

Ly49H 3D10 Thermo Fisher Scientific 13-5886-81, 50-4875-80

Mouse IgG1, Kappa Thermo Fisher Scientific 50-4714-80

MULT1 5D10 Thermo Fisher Scientific 12-5863-81, 14-5863-82

NK1.1 PK136 BioLegend 108720, 108722, 108745

NKG2A 16a11 Thermo Fisher Scientific 12-5897-82

NKG2D CX5 Thermo Fisher Scientific 25-5882-82

PD1 RPM1-30 Thermo Fisher Scientific 17-9981-80

PD1 29F.1A12 BioLegend 135220

Rabbit IgG Polyclonal Abcam Ab171870

Rabbit IgG XP® DA1E Cell signaling Technology 3900S

Rae1d RD-41 Thermo Fisher Scientific 12-5756-82

Rat IgG1, Kappa RTK2071 BioLegend 400418, 400443

Streptavidin Thermo Fisher Scientific, BL

46-4317-82, 405229

TCRb H57-597 BioLegend 109243, 109226

Thy1.2 30-H12 BioLegend 105324, 105331

TIGIT 1G9 BioLegend 142105

Tim3 RMT3-23 BioLegend 119706, 119715

TRAIL N2B2 BioLegend 109303

Armenian Hamster IgG Thermo Fisher Scientific 13-4113-85

Supplemental Table 1. Antibodies for flow cytometry. The table lists all antibodies used in this study,

including clone (when applicable), provider and catalogue number.

A

B

IL-15 In Vivo Model

Control

-15 -10 -5 0

Acute

Resolved

Chronic

Analysis

IL-15/IL15R

PBS

Days

IL-2 or Poly I:C In Vivo Model

Control

-15 -10 -5 0

Acute

Resolved

Chronic

Analysis

IL-2 or Poly I:C

PBS

Days

Supplemental Figure 1. Schematic representation of the NK cell stimulation models. In

order to study the impact of in vivo sustained stimulation, mice received a variety of known NK

cell activation reagents: IL-15/IL-15R (2.5μg/3μg), IL-2 (5x105 IU) and the TLR3 ligand poly I:C

(200μg). Chronic stimulation was achieved by continues i.p injections of the activation reagent at

the indicated days for each model. Acute and resolved stimulation was induced by i.p injections

of a given reagent during the early or late stage of the dosage regimen at the indicated days

respectively. 200ul of Phosphate buffered saline (PBS) was given at the indicated times as

control. (A-B) Dose regimen representation for the IL-15 (A) and the IL-2 and Poly I:C (B)

stimulation models.

*

*** *** ***

*** *** ***

* ****

*** * *** *

*** *** **

** **

**

**** **

*

*** **

* *** **

***** * *** *

** * ***

* ** *

***** *** *** ***

*** *** ** ***

*** *** *** ** *

*** *** ***

* *

IL-2 Poly I:CIL-2 Poly I:C

IL-2 Poly I:C

A B

C

Supplemental Figure 2. Exhaustion arises after prolonged IL-2 and Poly I:C stimulation. C57BL/6 mice

were treated with acute or chronic IL-2 or Poly I:C. Splenocytes were collected 24h after last treatment and

evaluated for NK cell phenotype and function. (A) Multivarian hetmap analysis for IL-2 and poly I:C models. (B)

Percentage of IFN after 4h NK.1.1 stimulation is shown for gated NK cells. (C) Percentage of tumor cell lysis is

shown. (D) Levels of IFNγ, TNFα, IL-12, GM-CSF, CCL2, CCL3, CXCL9, CXCL10 found in the serum of treated

mice. Data are representative of at least three independent experiments with an n=3 (mean ± SEM). One-way

ANOVA or two-way ANOVA (C) was done to assess significance (*p<0.05, **p<0.01, ***p<0.001). Statistics are

represented against the Acute treated group. No significant difference are found between the other groups

unless it is indicated.

0 1 2 3

0

2 0

4 0

6 0

8 0

1 0 0

* * *

* * *

*

**

E :T

% L

ys

is

Contr

ol

Acute

Resolv

ed

Chro

nic

0

5

1 0

1 5

%IF

N

* ** ** * *

Contr

ol

Acute

Resolv

ed

Chro

nic

0

5

1 0

1 5

2 0

2 5

* * ** * *

%IF

N

* * *

0 1 2 3

0

2 0

4 0

6 0

8 0

1 0 0C o n tro l

R eso lved

A c u te

C h ro n ic

* *

E :T

% L

ys

is

g

/mL

IF

N

IL - 1 5 IL - 2 Po ly I:C

0

2 0 0

4 0 0

6 0 0

8 0 0

C o n tro l

A c u te

C h ro n ic

******** **

g

/mL

IL

-12

IL - 1 5 IL - 2 Po ly I:C

0

1 0 0

2 0 0

3 0 0

4 0 0

C o n tro l

A c u te

C h ro n ic

***********

***

***

g

/mL

GM

-CS

F

IL - 1 5 IL - 2 Po ly I:C

0

5

1 0

1 5

2 0

C o n tro l

A c u te

C h ro n ic*

g

/mL

CC

L2

IL - 1 5 IL - 2 Po ly I:C

0

2 0 0

4 0 0

6 0 0

C o n tro l

A c u te

C h ro n ic

***

***

***

**

** **

g

/mL

CC

L3

IL - 1 5 IL - 2 Po ly I:C

0

1 0

2 0

3 0

4 0

5 0

C o n tro l

A c u te

C h ro n ic

g

/mL

CX

CL

10

IL - 1 5 IL - 2 Po ly I:C

0

2 0 0

4 0 0

6 0 0

8 0 0

C o n tro l

A c u te

C h ro n ic

******

***

**

**

*

g

/mL

TN

F

IL - 1 5 IL - 2 Po ly I:C

0

5 0

1 0 0

1 5 0

2 0 0

C o n tro l

A c u te

C h ro n ic

***

***

**

* **

**

g

/mL

CX

CL

9

IL - 1 5 IL - 2 Po ly I:C

0

5 0 0 0

1 0 0 0 0

1 5 0 0 0

C o n tro l

A c u te

C h ro n ic***

*** ****** ***

***

g

/mL

GM

-CS

F

IL - 1 5 IL - 2 Po ly I:C

0

5

1 0

1 5

2 0

C o n tro l

A c u te

C h ro n ic*

D

AControl ResolvedAcute Chronic

CD11b

CD27

Spleen

BM

IL-15B

C

Supplemental Figure 3. Impact of chronic stimulation on NK cell homeostasis. (A) Total

number of NK cells gated as CD3-NK1.1+ is displayed for IL-15, IL-2 and Poly I:C stimulation models.

(B) Representative dot-plots of immature-like (CD27-CD1b+) and mature-like (CD27+/-CD11b+) cells

of gated CD3-NK1.1+ NK cells is shown for spleen and BM in the IL-15 stimulation model. (C)

Change of MFI for the IL2R (CD122) expression after chronic stimulation on gated NK cells is

shown. Data represents 3 independent experiments with 3 mice per group (mean ± SEM). Significant

differences are displayed for comparisons with acutely stimulated groups (*p<0.05, **p<0.01,

***p<0.001). No significant differences were found between the other groups.

IL -1 5 IL -2 P o ly I:C

0

1 .51 0 6

3 .01 0 6

4 .51 0 6

C o n tro l

R eso lved

A c u te

C h ro n ic

****

******

***

***

***

S tim u la tio n

# N

K c

ell

s

(CD

3- N

K1

.1+

)

IL 2 R

IL 1 5 IL 2 P o ly I:C

0 .0

0 .1

0 .2

0 .3

0 .4

0 .5

0 .6

0 .7

0 .8

0 .9

1 .0

1 .1

C o n tro l

A c u te

C h ro n ic

R eso lved

Fo

ld C

ha

ng

e

MF

I

T im 3

IL 1 5 IL 2 P o ly IC

0

1 0

2 0

3 0

C o n tro l

A c u te

C h ro n ic

***

***

** *

***

***

%

P D 1

IL 1 5 IL 2 P o ly IC

0

1 0

2 0

3 0

C o n tro l

A c u te

C h ro n ic

%

T IG IT

IL 1 5 IL 2 P o ly I:C

0

1 0

2 0

3 0

4 0

C o n tro l

A c u te

C h ro n ic

*****

******

%

** *

T IG IT

IL 1 5 IL 2 P o ly I:C

0

5 0

1 0 0

1 5 0

2 0 0

2 5 0

C o n tro l

A c u te

C h ro n ic

MF

I

N K G 2 D

IL 1 5 IL 2 P o ly I:C

0

2 5

5 0

7 5

1 0 0

C o n tro l

A c u te

C h ro n ic

*******

*****

%

N K G 2 D

IL 1 5 IL 2 P o ly I:C

0

2 0 0

4 0 0

6 0 0

8 0 0

1 0 0 0

C o n tro l

A c u te

C h ro n ic

** *****

*

******

MF

I

N K G 2 D

IL 1 5 IL 2 P o ly I:C

0

2 0 0

4 0 0

6 0 0

8 0 0

1 0 0 0

C o n tro l

A c u te

C h ro n ic

** *****

*

******

MF

I

A

D

B

C

E

F

G

H

APC anti-mouse TIGIT

APC anti-mouse Tim3

APC anti-mouse PD1

PECy7 anti-mouse NKG2D

FMO Control Acute Chronic

Supplemental Figure 4. Impact of chronic stimulation on checkpoint inhibitors NK cell receptors. (A-B)

Representative dot plots (A), total percentage and MFI expression (B) of TIGIT on gated NK cells are shown. (C-D)

Representative dot plots (C) and total percentage (D) of Tim3 on gated NK cells are shown. (E-F) Representative dot plots

(E) and total percentage (F) of PD1 on gated NK cells are shown. (G-H) Representative dot plots (G), total Percentage and

MFI expression (H) of NKG2D on gated NK cells are shown. Data represents at least 3 independent experiments with 3 mice

per group (mean ± SEM). Significant differences are displayed for comparisons with acutely stimulated groups (*p<0.05,

**p<0.01, ***p<0.001). No significant differences were found between the other groups.

0 7 1 4 2 1 2 8

0

5

1 0

1 5

2 0

S a liv a ry G la n d

S p le e n

***

**

***

D a y s o f in fe c tio n

%

PD

1

PE

ant

i-mou

seN

KG

2A

FITC anti-mouse Ly49G2

M C M V v ira l tite rs

0 7 1 4 2 1 2 8

0

2

4

6

8

S p le e n

S a liv a ry G la n d

D a y s o f in fe c tio n

IE1

ge

ne

(lo

g10

co

pie

s/o

rga

n)

M C M V v ira l tite rs

0 7 1 4 2 1 2 8

0

2

4

6

8

S p le e n

S a liv a ry G la n d

D a y s o f in fe c tio n

IE1

ge

ne

(lo

g10

co

pie

s/o

rga

n)

D a y s o f in fe c tio n

%

Ly

49

H

0 7 1 4 2 1 2 8

0

2 0

4 0

6 0

8 0

1 0 0

S a liv a ry G la n d

S p le e n

BV605 anti-mouse Ly49H

PB

anti-m

ouse C

D27 Spleen Salivary Gland

0 7 1 4 2 1 2 8

0

2 0

4 0

6 0

8 0

1 0 0S a liv a ry G la n d

S p le e n** ***

D a y s o f in fe c tio n

% N

KG

2A

D a y s o f in fe c tio n

%IF

N

0 7 1 4 2 1 2 8

0

2 0

4 0

6 0

8 0

1 0 0

L y 4 9 H+

L y 4 9 H-

***

**

D a y s o f in fe c tio n

%

Ki6

7

0 7 1 4 2 1 2 8

0

1 0

2 0

3 0

4 0

5 0

L y 4 9 H+

L y 4 9 H-

***

D a y s o f in fe c tio n

MF

I E

om

es

0 7 1 4 2 1 2 8

1 0 0 0

1 5 0 0

2 0 0 0

2 5 0 0

L y 4 9 H+

L y 4 9 H-

***

**

** ** ** **

D a y s o f in fe c tio n

MF

I N

KG

2D

0 7 1 4 2 1 2 8

1 5 0

2 0 0

2 5 0

3 0 0

3 5 0

L y 4 9 H+

L y 4 9 H-

***

*

*** *****

D a y s o f in fe c tio n

%

KL

RG

1

0 7 1 4 2 1 2 8

0

2 0

4 0

6 0

8 0

1 0 0

L y 4 9 H+

L y 4 9 H-

***

*** *** ******

D a y s o f in fe c tio n

% G

ran

B

0 7 1 4 2 1 2 8

0

2 0

4 0

6 0

8 0

1 0 0

L y 4 9 H+

L y 4 9 H-

***

***

*** *****

A

E F G

H I J K L M

B

DC

Supplemental Figure 5. Expression of NK cell markers after chronic MCMV infection. (A) Representative dot plots

of the inhibitory receptors NKG2A and Ly49G2 is shown for gated NK cells derived from the spleen (black) or the salivary

gland (blue) on MCMV infected mice. (B) Total percentage of NKG2A expression on gated NK cells is shown. (C-D)

Representative dot plots (C) and total percentage (D) of PD1 and Tim3 on gated NK cells are shown. (E) MCMV viral

titers (IE1 gene copies) of spleen and salivary gland at different time points after MCMV infection. (F) Representative dot-

plots of CD27 and Ly49H expression from the spleen and salivary gland on non-infected mice (G) Total percentage of

activation marker Ly49H on gated NK cells at different time points of MCMV infection on NK cells (CD3-NK1.1+). (H-M)

Total percentage or MFI of Ki67 (H), IFNγ (I), Granzyme B (J), Eomes (K), NKG2D (L) and KLRG1 (M) on Ly49H-

(square symbol) or Ly49H+ (circle symbol) NK cells is shown from the spleen of infected MCMV mice. IFNγ was

measure after NK1.1 stimulation. Data represents three independent experiments with 4-5 mice per group except in

panel E which was done one time (mean ± SEM). Significant differences are displayed for comparisons with non-infected

controls at day 0. One-way ANOVA was used to asses(*p<0.05, **p<0.01, ***p<0.001).

Day 0 Day 3 Day 7 Day 14 Day 21 Day 28

SG

Spleen

D a y s o f in fe c tio n

%

KL

RG

1

0 7 1 4 2 1 2 8

0

2 0

4 0

6 0

8 0

1 0 0

L y 4 9 H+

L y 4 9 H-

***

*** *** ******

D a y s o f in fe c tio n

%

KL

RG

1

0 7 1 4 2 1 2 8

0

2 0

4 0

6 0

8 0

1 0 0

L y 4 9 H+

L y 4 9 H-

***

*** *** ******

PE

Cy7

ant

i-mou

seT

im3

BV605 anti-mouse PD1

0 7 1 4 2 1 2 8

0

2

4

6

S a liv a ry G la n d

S p le e n

D a y s o f in fe c tio n

% T

im3

* **

**S

GS

pleen

0 5 1 0 1 5 2 0

0

2 0

4 0

6 0

8 0

1 0 0

0

4

7

9

*** ***

***

******

*

*

*

**

**

E :T

% L

ys

is

AF660 anti-mouse Eomes

PE

Cy7

ant

i-mou

seT

-bet

PECy7 anti-mouse NKG2D

PB

ant

i-mou

se T

hy1.

2

FITC anti-mouse Ly49G2

PE

ant

i-mou

se N

KG

2A

Day 0 Day 4 Day 7 Day 9A

B

C

D

E F

G H I

d a y s p o s t-a c t iv a t io n

MF

I T

hy

1.2

0 2 4 6 8 1 0

0

5 0 0 0

1 0 0 0 0

1 5 0 0 0

* * * * * * * *

* * * *

d a y s p o s t-a c t iv a t io n

MF

I L

y4

9G

2

0 2 4 6 8 1 0

0

2 0 0 0

4 0 0 0

6 0 0 0

8 0 0 0

* * * * * * * * *

* * * * * *

d a y s p o s t-a c t iv a t io n

% N

K c

ell

s

0 2 4 6 8 1 0

0

2 0

4 0

6 0

8 0

1 0 0

E o m e s

T bet

N K G 2D

T h y 1 .2

L y 4 9 G 2

N K G 2A

K L R G 1

K i6 7

Supplemental Figure 6. Long-term in vitro IL-2 stimulation recapitulates NCE phenotype. Thy1.2- cells

were cultured with IL-2 and adherent NK cells were collected and analyzed for NCE phenotype and function at

different time points (control unstimulated: day 0; acutely stimulation: day 4; chronic stimulation: days 7 and 9).

(A) Representative dot plots of Eomes and T-bet expression on gated NK cells (CD3-NK1.1+) is shown at

different time points of stimulation. (B) Representative dot plots of NKG2D and Thy1.2 is shown for gated NK

cells at different time points of stimulation. (C) Representative dot plots of the inhibitory receptors Ly49G2 and

NKG2A is shown for gated NK cells at different time points of stimulation. (D) Total percentage of NK cell

markers is shown at different time points of stimulation on gated NK cells. (E-F) MFI expression for Thy1.2 (E)

and Ly49G2 (F) is shown for gated NK cells. (G) Percentage of cells expressing Ki67 on NK cells. (H-I) The

percentage of IFNγ and TNFα producing NK cells after NK1.1 (H) or CD16 stimulation (I). (J) Total percentage of

tumor lysis is shown at different time points of activation. Data are representative of at least three independent

experiments done by triplicate (mean ± SEM). One-way ANOVA (D-I) or Two-way ANOVA (J) was done to

assess significance (*p<0.05, **p<0.01, ***p<0.001), which comparison was made against the peak of activation

on day 4 (acute stimulation). No differences between the other groups were found unless otherwise indicated.

N K 1 .1

d a y s p o s t-a c tiv a tio n

% N

K c

ell

s

0 2 4 6 8 1 0

0

1 0

2 0

3 0

4 0

IF N

TNF

* * * * * * * * *

* *

* * * * * * * * *

* * *

* * ** * * * * *

n .s .

C D 1 6

d a y s p o s t-a c tiv a tio n

% N

K c

ell

s

0 2 4 6 8 1 0

0

5

1 0

1 5

2 0

IF N

TNF

* * * * * * * * *

* * * * * * * *

* *

* * ** * * * * *

* * *

n .s .

Ki67

0 2 4 6 8 100

20

40

60

80

100

***

***

***

%

days post-activation

%

J

Nu

mb

er

of

NK

ce

lls

Contr

ol

Acute

Chro

nic

0

1 .01 0 6

2 .01 0 6

3 .01 0 6

4 .01 0 6

IgG

a n ti-N K G 2D

*

Nu

mb

er

of

NK

ce

lls

Contr

ol

Acute

Chro

nic

0

1 .01 0 6

2 .01 0 6

3 .01 0 6

4 .01 0 6

5 .01 0 6

W T

N KG 2D KO*

% D

NM

A1

Contr

ol

Acute

Chro

nic

0

2 0

4 0

6 0W T

N KG 2D KO

% T

RA

IL

Contr

ol

Acute

Chro

nic

0

2 0

4 0

6 0W T

N KG 2D KO

% F

as

L

Contr

ol

Acute

Chro

nic

0

5

1 0

1 5

2 0W T

N KG 2D KO

% T

IGIT

Contr

ol

Acute

Chro

nic

0

1 0

2 0

3 0

4 0W T

N KG 2D KO

% C

D9

6

Contr

ol

Acute

Chro

nic

0

2 0

4 0

6 0

8 0

1 0 0W T

N KG 2D KO

% N

KG

2A

Contr

ol

Acute

Chro

nic

0

2 0

4 0

6 0

8 0

1 0 0W T

N KG 2D KO

*

% D

NM

A1

Contr

ol

Acute

Chro

nic

0

2 0

4 0

6 0

8 0

IgG

a n ti-N K G 2D

% T

RA

IL

Contr

ol

Acute

Chro

nic

0

2 0

4 0

6 0

IgG

a n ti-N K G 2D

% C

D9

6

Contr

ol

Acute

Chro

nic

0

2 0

4 0

6 0

8 0

1 0 0

IgG

a n ti-N K G 2D

% T

IGIT

Contr

ol

Acute

Chro

nic

0

1 0

2 0

3 0

4 0

5 0

IgG

a n ti-N K G 2D%

Fa

sL

Contr

ol

Acute

Chro

nic

0

1 0

2 0

3 0

4 0

IgG

a n ti-N K G 2D

% N

KG

2A

Contr

ol

Acute

Chro

nic

0

2 0

4 0

6 0

8 0

IgG

a n ti-N K G 2D

A

B

C

E

g

/mL

IF

N

Contr

ol

Acute

Chro

nic

0

2 0 0

4 0 0

6 0 0

8 0 0

W T

a n ti-N K G 2D

***

g

/mL

TN

F

Contr

ol

Acute

Chro

nic

0

1 0 0

2 0 0

3 0 0

4 0 0

W T

a n ti-N K G 2D

***

***

***

g

/mL

IL

-12

Contr

ol

Acute

Chro

nic

0

5 0

1 0 0

1 5 0

W T

a n ti-N K G 2D

**

g

/mL

GM

-CS

F

Contr

ol

Acute

Chro

nic

0

5

1 0

1 5

2 0

W T

a n ti-N K G 2D

g

/mL

CC

L2

Contr

ol

Acute

Chro

nic

0

2 0 0

4 0 0

6 0 0

W T

a n ti-N K G 2D

***

*

g

/mL

CC

L3

Contr

ol

Acute

Chro

nic

0

1 0 0

2 0 0

3 0 0

4 0 0

W T

a n ti-N K G 2D

****

g

/mL

CX

CL

9

Contr

ol

Acute

Chro

nic

0

4 0 0 0

8 0 0 0

1 2 0 0 0

W T

a n ti-N K G 2 D

* * *

g

/mL

CX

CL

10

Contr

ol

Acute

Chro

nic

0

2 0 0

4 0 0

6 0 0

8 0 0

W T

a n ti-N K G 2D

******

g

/mL

GM

-CS

F

Contr

ol

Acute

Chro

nic

0

5

1 0

1 5

2 0

W T

a n ti-N K G 2D

g

/mL

GM

-CS

F

Contr

ol

Acute

Chro

nic

0

5

1 0

1 5

2 0

W T

a n ti-N K G 2D

Supplemental Figure 7. Impact of NKG2D deficiency on NK cells. (A) Total number of NK cells is shown for in vivo

IL-2 stimulated mouse where NKG2D was absence (upper level) or blockade (lower level) at the time of stimulation. (B)

Percentage of NK cell activating receptors DNAM1, TRAIL and FasL gated on NK cells is shown for IL-2 treated WT and

NKG2D KO mice (upper level) or control or anti-NKG2D mice (lower level). (C) Total number of CD3+CD8+ T cells is

shown L-2 treated WT and NKG2D KO mice (upper level) or control or anti-NKG2D mice (lower level). (D) Percentage

of NK cell inhibitory receptors CD96, TIGIT and NKG2A gated on NK cells is shown L-2 treated WT and NKG2D KO

mice (upper level) or control or anti-NKG2D mice (lower level).(E) Levels of IFNγ, TNFα, IL-12, GM-CSF, CCL2, CCL3,

CXCL9, CXCL10 found in the serum of treated mice. Data represents at least 3 independent experiments (mean ± SEM).

Significant differences are displayed for comparisons with WT and NKG2D deficient/blockade groups (*p<0.05, **p<0.01,

***p<0.001).

Nu

mb

er

of

CD

8+

T c

ells

Contr

ol

Acute

Chro

nic

0

5 .01 0 6

1 .01 0 7

1 .51 0 7

2 .01 0 7

2 .51 0 7

W T

N KG 2D KO

Nu

mb

er

of

CD

8+

T c

ells

Contr

ol

Acute

Chro

nic

0

5 .01 0 6

1 .01 0 7

1 .51 0 7

IgG

a n ti-N K G 2D

D

A B

Supplemental Figure 8. Long-term in vitro NK cell activation on NKG2D deficient mice has a

reduced exhaustion phenotype. (A) Percentage of IFNγ production upon NK1.1 stimulation and tumor

lysis at 10:1 E:T ratio for IL-2 activated WT and NKG2D KO NK cells. (B) Percentage of Ki67 on gated NK

cells. (C) MFI or percentage of Eomes, Ly49G2, Thy1.2 and KLRG1 are shown for WT and NKG2D KO NK

cells. Data are representative of three experiments performed in triplicate (mean ± SEM). Two-Way

ANOVA was used to assess significance. Significant differences are displayed for comparisons between

WT and NKG2D KO at each time point (*p<0.05, **p<0.01, ***p<0.001)

E :T 10 :1

WT

NK

G2D

KO

0

2 0

4 0

6 0

8 0

1 0 0

4

7

9

* ***

% L

ys

is

D a y s p o s t-a c tiv a tio n

0 2 4 6 8 1 0

0

2 0

4 0

6 0

8 0

1 0 0

W T

N KG 2D KO

***

D a y s p o s t-a c tiv a tio n

% K

i67

0 2 4 6 8 1 0

0

1 5 0 0

3 0 0 0

4 5 0 0

6 0 0 0

7 5 0 0

9 0 0 0

W T

N KG 2D KO

D a y s p o s t-a c tiv a tio n

MF

I E

om

es

0 2 4 6 8 1 0

0

1 7 5 0

3 5 0 0

5 2 5 0

7 0 0 0

W T

N KG 2D KO

D a y s p o s t-a c tiv a tio n

MF

I L

y4

9G

2

0 2 4 6 8 1 0

0

4 0 0 0

8 0 0 0

1 2 0 0 0

W T

N KG 2D KO

******

D a y s p o s t-a c tiv a tio n

MF

I T

hy

1.2

0 2 4 6 8 1 0

0

2 5

5 0

7 5

W T

N KG 2D KO

*

D a y s p o s t-a c tiv a tio n

% K

LR

G1

0 2 4 6 8 1 0

0

1 0

2 0

3 0

4 0

W T

N KG 2D KO

D a y s p o s t-a c tiv a tio n

% I

FN

0 2 4 6 8 1 0

0

2 0

4 0

6 0

8 0

1 0 0

W T

N KG 2D KO

***

D a y s p o s t-a c tiv a tio n

% K

i67

D

C

A B

CD45 NKG2D Merged

Day 9

MULT1

Day 9 KU

C D

Supplemental Figure 9. Impact of KU on NK cells during in vitro activation. (A) The percentage

of phosphorylation of the ATM-dependent protein Chk2 (pChk2) on gated NK cells. (B) Changes in

the level of expression of MULT1 (percentage, MFI and mRNA) is shown for NK cells after KU

treatment. (C) Representative immunofluorescence images of day 9 IL-2 in vitro stimulated NK cells

after KU treatment showing CD45, NKG2D and MULT1 expression. (D) Total percentage of NKG2D

internalization observed in immunofluorescence images in control or KU-treated NK cells collected on

day 9. Data are representative of at lest 3 independent experiments done in triplicate (mean ± SEM).

One-way ANOVA (A-B) or T-test study (B) were used to assess the significance between control and

KU-treated NK cells (*p<0.05, **p<0.01, ***p<0.001).

% M

UL

T1

0 4 7 7 9 9

0

1 0

2 0

3 0 ***

***

D a y s :

K U : - - - + - +

% p

CH

K2

0 4 7 9 9

0

2 0

4 0

6 0 ***

*

D a y s :

K U : - - - + - +

* ***

***

**

**

Fo

ld C

ha

ng

e

MU

LT

1 M

FI

vs

Da

y 0

0 4 7 7 9 9

0

1

2

3

4 ***

* *** ****

D a y s :

K U : - - - + - +

Fo

ld C

ha

ng

e M

UL

T1

mR

NA

ex

pre

ss

ion

vs

da

y 0

0 4 9 9

0

5

1 0

1 5

D a y s :

K U : - - - +

**

*

Co n tr o l K U

0

1 0

2 0

3 0

4 0

***

% N

KG

2D

In

tern

aliz

ati

on

1.26

12.96

A

Da

y 0

Da

y 4

Da

y 9

Da

y 9

KU

Upregulated Genes

B C

Supplemental Figure 10. Molecular changes induced during long-term in vitro IL-2 activation.

(A) Heatmap representation of the gene expression profile of RNA obtained from freshly isolated NK

cells (day 0) or NK cells collected at the peak of activation on day 4 or after exhaustion on day 9

during in vitro IL-2 stimulation with or without KU treatment (KU day 9). (B) Microarray analysis

showing the overlap of up-regulated genes with significant differential expression on aNK cells at

different time points when compared to gene expression of fresh NK cells (day 0). (C) Predicted

biological processes affected by the up-regulated genes after IPA analysis is shown. Data are

representative of 2 independent experiments done in duplicate (F-G). Dotted red lines indicate z-

score value below 2.

P re d ic tio n o f B io lo g ic a l P ro c e s s

- 2 .5 0 .0 2 .5 5 .0 7 .5 1 0 .0

Ty r o s in e Ph o s p h o r y la tio n

Ph o s p h o r y la tio n o f Pr o te in

L y mp h o c y te Ho me o s ta s is

Ce ll Pr o lif e r a tio n

Ce ll S u r v iv a l

Cy to to x ic ity o f NK c e lls

Ce ll De a th

A p o p to s is

D a y 9 K U

D a y 9

D a y 4

n .d

A c tiv a tio n z -s c o re

0 3 7 11Day

Fresh

NK

Control

aNK

KU

aNK

Supplemental Figure 11. In vivo proliferation and expansion of activated NK cells after

adoptive transfer. C57BL/6 L2G85 Luc+ fresh NK cells or ex vivo IL-2 stimulated NK cells (control

aNK or KU treated) were transferred into BALB/c Rag2-/-IL2Rc-/- deficient mice and treated with

5x104 IU IL-2 for 7 days. NK cell proliferation, expansion and survival was measured by

bioluminescence at different time points. (A) Representative images of bioluminescence signal at

different time points for the different groups. (B) Graphical representation of the bioluminescence

image. Data is representative of two independent experiments with 3 mice per group (mean ± SEM).

One-way ANOVA was done to assess the significance (*p<0.05, **p<0.01, ***p<0.001).

0 2 4 6 8 1 01 2

0

5 .01 0 6

1 .01 0 7

1 .51 0 7

F re sh N K

C o n tro l a N K

K U aNK

D a y s a f te r tra n s fe r

Ra

dia

nc

e

(p/s

c/c

m2/s

r)

*****

***

*

***

****