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)
*****
***
*
***
****