Supplementary Materials for - Science · 2020. 3. 16. · 3 Fig. S5. Systemic T cell–independent...
16
immunology.sciencemag.org/cgi/content/full/5/45/eaaz2738/DC1 Supplementary Materials for NK cells mediate clearance of CD8 + T cell–resistant tumors in response to STING agonists Christopher J. Nicolai, Natalie Wolf, I-Chang Chang, Georgia Kirn, Assaf Marcus, Chudi O. Ndubaku, Sarah M. McWhirter, David H. Raulet* *Corresponding author. Email: [email protected] Published 20 March 2020, Sci. Immunol. 5, eaaz2738 (2020) DOI: 10.1126/sciimmunol.aaz2738 The PDF file includes: Fig. S1. MHC I expression and growth of B2m –/– tumor cell lines. Fig. S2. Verifying in vivo depletions. Fig. S3. Representative flow plots for Fig. 3A. Fig. S4. Representative flow plots for Fig. 3B. Fig. S5. Systemic T cell–independent antitumor effects of CDNs in B16-F10-B2m –/– . Fig. S6. Representative flow plots for Fig. 4B. Fig. S7. IFNAR1 neutralization prevents CDN-induced NK cell activation, cytotoxicity, and tumor rejection. Fig. S8. Bone marrow chimera reconstitution efficiency. Fig. S9. NK cell and T cell IFNAR1 expression in Ncr1-iCre, Ifnar1 fl/fl mice. Fig. S10. Representative flow plots for Fig. 5A. Fig. S11. IFNAR1 expression by DCs and NK cells in CD11c-Cre, Ifnar1 fl/fl mice. Fig. S12. Representative flow plots for Fig. 6B. Fig. S13. IFNAR1 neutralization reduces CDN-induced IL-15RA expression. Fig. S14. Representative flow plots for Fig. 7C. Other Supplementary Material for this manuscript includes the following: (available at immunology.sciencemag.org/cgi/content/full/5/45/eaaz2738/DC1) Table S1. Raw data file (in Excel spreadsheet).
Transcript of Supplementary Materials for - Science · 2020. 3. 16. · 3 Fig. S5. Systemic T cell–independent...
immunology.sciencemag.org/cgi/content/full/5/45/eaaz2738/DC1
Supplementary Materials for
NK cells mediate clearance of CD8+ T cell–resistant tumors in response
to STING agonists
Christopher J. Nicolai, Natalie Wolf, I-Chang Chang, Georgia Kirn, Assaf Marcus, Chudi O. Ndubaku, Sarah M. McWhirter, David H. Raulet*
*Corresponding author. Email: [email protected]
Published 20 March 2020, Sci. Immunol. 5, eaaz2738 (2020)
DOI: 10.1126/sciimmunol.aaz2738
The PDF file includes:
Fig. S1. MHC I expression and growth of B2m–/– tumor cell lines. Fig. S2. Verifying in vivo depletions. Fig. S3. Representative flow plots for Fig. 3A. Fig. S4. Representative flow plots for Fig. 3B. Fig. S5. Systemic T cell–independent antitumor effects of CDNs in B16-F10-B2m–/–. Fig. S6. Representative flow plots for Fig. 4B. Fig. S7. IFNAR1 neutralization prevents CDN-induced NK cell activation, cytotoxicity, and tumor rejection. Fig. S8. Bone marrow chimera reconstitution efficiency. Fig. S9. NK cell and T cell IFNAR1 expression in Ncr1-iCre, Ifnar1fl/fl mice. Fig. S10. Representative flow plots for Fig. 5A. Fig. S11. IFNAR1 expression by DCs and NK cells in CD11c-Cre, Ifnar1fl/fl mice. Fig. S12. Representative flow plots for Fig. 6B. Fig. S13. IFNAR1 neutralization reduces CDN-induced IL-15RA expression. Fig. S14. Representative flow plots for Fig. 7C.
Other Supplementary Material for this manuscript includes the following: (available at immunology.sciencemag.org/cgi/content/full/5/45/eaaz2738/DC1)
Table S1. Raw data file (in Excel spreadsheet).
Fig. S1. MHC I expression and growth of B2m–/– tumor cell lines. (A) WT and B2m-/- tumor cells were
stained with anti-MHC class I (H-2Kb clone AF6-88.5 for C1498 and B16-F10 and H-2Kd clone SF1-1.1
for CT26 and 4T1) or isotype control. For B16-F10, some cells were incubated with 100 ng/ml IFN-
(Biolegend) for 24 hours before staining. (B) CDN-induced delay in tumor growth in Rag-/-Il2rg-/- mice
are TNF--dependent. RMA-B2m-/- tumors were established in Rag2-/Il2rg-/- mice, treated and analyzed
as in Fig. 1B. Some mice were given TNF- neutralizing antibody or control Ig (see Methods). n=3-4 per
group. Representative of two independent experiments.
Nor
mal
ized
to M
ode100
80
60
40
20
0 103 104 105
WTB2m-/-
isotype
+ IFN-
Nor
mal
ized
to M
ode100
80
60
40
20
0 103 104 105
WTB2m-/-
isotype
B16-F10
Nor
mal
ized
to M
ode
100
80
60
40
20
0 103 104 105
WTB2m-/-
isotype
CT26
Nor
mal
ized
to M
ode100
80
60
40
20
0 103 104 105
WTB2m-/-
isotype
4T1
Nor
mal
ized
to M
ode
100
80
60
40
20
0 103 104 105
WTB2m-/-
isotype
C1498
H2-Kb
H2-Kd
Rag2-/-Il2rg-/- mice
-5 0 5 10 15 200
300
600
900
1200
Day
tum
or v
olum
e (m
m3 )
PBS
CDN + Ctrl Ig
CDN + TNF-ab
p=0.0083CDN
ns
A
B
Fig. S2. Verifying in vivo depletions. (A) Success of T cell depletion protocols. Mice were treated with
control Ig (left panel), CD4 T cell-depleting antibodies (center panel) or CD8 T cell-depleting antibodies
(left panel) at Day= -2 and Day= -1, as described in methods. On Day=0, Blood was collected, ACK-
treated, and stained for viable, CD3+ cells. (B) Success of NK cell depletion protocols. Mice were either
untreated (left panel) or NK cell-depleted (left panel) at Day= -2 and Day= -1, as described in methods.
On Day=0, splenocytes were collected, ACK-treated, and gated on viable, CD45+, CD3-, CD19- cells.
2.7% 0.06%
NKp46
NK
1.1
103 104 1050
104
105
0
104
105
0
103 104 1050
Untreated +NK1.1 ab
CD4
CD
8
Ctrl Ig CD4-depleted CD8-depleted
43.4%
5.9% 50.4%
87.3%
12.6% 0.08% 7.5% 91.1%
1.0%
A
B
Fig. S3. Representative flow plots for Fig. 3A.
Fig. S4. Representative flow plots for Fig. 3B.
Fig. S5. Systemic T cell–independent antitumor effects of CDNs in B16-F10-B2m–/–. Mice were CD4- and
CD8-depleted and B16-F10-B2m-/- tumors were established in both flanks at different doses (4x106 cells for
treated and 2x106 cells for distal). 5 days later one tumor was treated with PBS or 50 g CDN and tumor growth
at both sites was monitored and analyzed as described in Fig. 1B. (n=6 per group). Two individual experiments
are shown.
-5 0 5 10 150
200
400
600
800
Day
tum
or v
olum
e (m
m3 ) Treated tumor
PBS
CDN
CDN/PBS
p<0.0001
-5 0 5 10 150
200
400
600
800
Day
tum
or v
olum
e (m
m3 ) Contralateral tumor
PBS
CDN
p<0.0001
-5 0 5 10 15 200
50100150200250
Day
tum
or v
olum
e (m
m3) Treated tumor
PBS
CDN
CDN/PBS p<0.0001
-5 0 5 10 150
50100150200250300
Daytu
mor
vol
ume
(mm
3 ) Contralateral tumorPBS
CDN
p=0.0005
Exp
erim
ent 1
Exp
erim
ent 2
Fig. S6. Representative flow plots for Fig. 4B.
WT
mic
e +
P
BS
WT
mic
e +
C
DN
Ifnar
1-/- m
ice
+ C
DN
%IFN- %CD107a+ GzmB MFI %Sca-1+
17.8%
54.9%
5.3%
10.6%
56.2%
8.1%
1,664
2,694
1,233
29.0%
92.9%
GzmB
NK
1.1 7.6%
Sca1
NK
1.1
CD107a
NK
1.1
IFN-
NK
1.1
Tumor-draining LN NK cells
103
103
103104 1050
103
104
105
0
103
103
103 103
103
103 103 1041050
103
104
105
0
103
103 1040
103
104
105
0
1041050
103
104
105
0
104 1050
103
104
105
0
104 1050
103
104
105
0
104 1050
103
104
105
0
104 1050
103
104
105
0
104 1050
103
104
105
0
104 1050
103
104
105
0
104 1050
103
104
105
0
104 1050
103
104
105
0
105
Fig. S7. IFNAR1 neutralization prevents CDN-induced NK cell activation, cytotoxicity, and tumor
rejection. (A and B) IFNAR1-neutralization prevents CDN-induced NK cell activation. RMA-B2m-/-
tumors were established and treated as described in Fig.1B. 24 h later tumor-draining lymph node cells
(A), and spleens (B) were harvested for flow cytometry analysis as in Fig. 3A. One group received
IFNAR1 neutralizing antibody or control Ig (see Methods). n=3-5. Data (representative of 2 independent
0 10 20 300
500
1000
1500
Day
tum
or v
olum
e (m
m3 ) RMA-B2m-/-
CDN
WT miceWT mice, Ifnar1-/- tumors
Ifnar1-/- miceIfnar1-/- mice, Ifnar1-/- tumors
*****
RMA-B2m-/-
-5 0 5 10 15 20 250
200400600800
1000 IFNAR1 ab
Ctrl Ig
p=0.002CDN
Day
tum
or v
olum
e (m
m3 )
25 50 100
200
0
5
10
15
E:T Ratio
%S
peci
fic L
ysis
WT + PBS
WT + CDN
WT + CDN + IFNAR1 ab
p<0.0001
C
E
D
PBSCDN
CDN + IF
NAR1 ab
01020304050
%IF
N-
+**** ****
ns
PBSCDN
CDN + IF
NAR1 ab
0
10
20
30
%C
D10
7a+ **** ****
ns
PBSCDN
CDN + IF
NAR1 ab
0
2000
4000
6000
8000
Gzm
B M
FI **** ****
ns
PBSCDN
CDN + IF
NAR1 ab
020406080
100
%S
ca-1
+
**** ****ns
Tumor-draining LN NK cellsP
BS
CD
NC
DN
+ IF
NA
R1
ab
%IFN- %CD107a+ GzmB MFI %Sca-1+
8.8%
40.5%
8.2%
7.4%
24.2%
7.6%
1,546
6,597
1,911
10.6%
79.6%
GzmB
NK
1.1 17.6%
Sca1
NK
1.1
CD107a
NK
1.1
IFN-
NK
1.1
103
103
103104 1050
103
104
105
0
103
103
103 103
103
103 103 1041050
103
104
105
0
103
103 104 1050
103
104
105
0
104 1050
103
104
105
0
104 1050
103
104
105
0
104 1050
103
104
105
0
104 1050
103
104
105
0
104 1050
103
104
105
0
1041050
103
104
105
0
104 1050
103
104
105
0
104 1050
103
104
105
0
104 1050
103
104
105
0
A
B
PBSCDN
CDN + IF
NAR1 ab
0
10
20
30
%IF
N-
+ **** ****ns
Splenic NK cells
PBSCDN
CDN + IF
NAR1 ab
0
2000
4000
6000
Gzm
B M
FI **** ****
*
PBSCDN
CDN + IF
NAR1 ab
0
20
40
60
80
%S
ca-1
+ **** ****ns
experiments) analyzed by one-way ANOVA with Tukey’s correction for multiple comparisons. *P < 0.05
****P<0.0001. (C) IFNAR1-neutralization reduces CDN-induced cytotoxicity. Cytotoxicity of
splenocytes from tumor-bearing PBS or CDN-treated mice analyzed as in Fig. 3C. Some mice injected
with IFNAR1 neutralizing antibody (see Methods). Data shown are technical replicates and representative
of two independent experiments. Error bars are shown but typically too small to see. (D) Host, and not
tumor, IFNAR1 is necessary for the antitumor effects of CDNs. RMA-B2m-/- Ifnar1-/- tumor cells were
generated using CRISPR-Cas9 and, along with RMA-B2m-/- tumors, were established, treated, and
analyzed as in Fig. 1B. **P<0.01; ***P < 0.001. Data combined from two independent experiments.
n=10 (E) IFNAR1-neutralization prevents CDN-induced tumor rejection. RMA-B2m-/- tumors were
established, treated, and analyzed as in Fig. 1B. Mice received IFNAR1 neutralizing antibody or control
rat Ig. Data are representative of two independent experiments. n=4-5.
Fig. S8. Bone marrow chimera reconstitution efficiency. Eight weeks after reconstitution, blood from
bone marrow chimeras was collected, treated with ACK, and stained for flow cytometry.
WT
WT
Ifnar
1-/-
WT
WT
Ifn
ar1-/-
Ifnar
1-/-
Ifnar
1-/-
0
20
40
60
80
100%
Pos
itive
CD45.1 (WT)
CD45.2 (Ifnar1-/-)
WT
WT
Ifnar
1-/-
WT
WT
Ifn
ar1-/-
Ifnar
1-/-
Ifnar
1-/-
0
1000
2000
3000
4000
IFN
AR
1 M
FI
Fig. S9. NK cell and T cell IFNAR1 expression in Ncr1-iCre, Ifnar1fl/fl mice. Representative
histograms of IFNAR1 expression on NK cells and T cells are shown along with average MFI. Blood
cells were ACK-treated. NK cells were gated as in Fig. 3A. T cells were gated as viable, CD19-, NK1.1-,
CD3+ cells. n=6-7. Though IFNAR staining intensity was decreased on both NK cells and T cells from
Ifnar1fl/fl mice compared to WT mice, we observed no functional defects in antitumor responses in those
mice.
0 103 104 105
Nor
mal
ized
to M
ode
20
40
60
80
100
WTIfnar1fl/fl
Ncr1-Cre Ifnar1fl/fl
Ifnar1-/-
NK cells
0 103 104 105
Nor
mal
ized
to M
ode
20
40
60
80
100
T cells
WT
Ifnar1fl/flIfnar1-/- Ncr1-Cre Ifnar1fl/fl
WT m
ice
Ifnar
1-/-
mice
Ifnar
1fl/f
l mice
Ncr1-
Cre If
nar1fl/f
l mice
0
2000
4000
6000
8000
10000
IFN
AR
1 M
FI NK cells
T cells
IFNAR1
Fig. S10. Representative flow plots for Fig. 5A.
PB
SC
DN
Ncr
1-C
re
+ C
DN
%IFN- %CD107a+ GzmB MFI %Sca-1+
4.9%
35.4%
19.0%
6.0%
24.7%
17.3%
1,414
4,201
2,494
7.8%
88.1%
GzmB
NK
1.1 43.9%
Sca1N
K1.
1CD107a
NK
1.1
IFN-
NK
1.1
Tumor-draining LN NK cells: all mice Ifnar1fl/fl
103
103
103 103
103
103 103
103
103 103
103
1031041050
103
104
105
0
1041050
103
104
105
0
1041050
103
104
105
0
1041050
103
104
105
0
1041050
103
104
105
0
1041050
103
104
105
0
1041050
103
104
105
0
1041050
103
104
105
0
1041050
103
104
105
0
104 1050
103
104
105
0
104 1050
103
104
105
0
104 1050
103
104
105
0
Fig. S11. IFNAR1 expression by DCs and NK cells in CD11c-Cre, Ifnar1fl/fl mice. Representative
histograms of IFNAR1 expression on DCs and NK cells. Splenocytes collected from the indicated
genotypes and ACK-treated. DCs and NK cells gated as in Fig. 7B and Fig. 3A, respectively.
0 103 104 105
Nor
mal
ized
to M
ode
20
40
60
80
100WTIfnar1fl/fl
CD11c-Cre Ifnar1fl/fl
Ifnar1-/-
Dendritic cells
0 103 104 105
Nor
mal
ized
to M
ode
20
40
60
80
100NK cells
WTIfnar1fl/fl
Ifnar1-/-CD11c-Cre Ifnar1fl/fl
IFNAR1
Fig. S12. Representative flow plots for Fig. 6B.
PB
SC
DN
CD
11c-
Cre
+
CD
N
%IFN- %CD107a+ GzmB MFI %Sca-1+
GzmB
NK
1.1
Sca1N
K1.
1CD107a
NK
1.1
IFN-
NK
1.1
Tumor-draining LN NK cells: all mice Ifnar1fl/fl
103
103
103 103
103
103 103
103
103 103
103
1031041050
103
104
105
0
1041050
103
104
105
0
1041050
103
104
105
0
1041050
103
104
105
0
1041050
103
104
105
0
1041050
103
104
105
0
1041050
103
104
105
0
1041050
103
104
105
0
1041050
103
104
105
0
104 1050
103
104
105
0
104 1050
103
104
105
0
104 1050
103
104
105
02,070
6,197
4,284
6.8%
32.2%
21.2%
5.5%
19.0%
14.7%
8.2%
79.8%
61.1%
Fig. S13. IFNAR1 neutralization reduces CDN-induced IL-15RA expression. RMA-B2m-/- tumors
were established and treated as in Fig. 1B. One group of CDN-treated mice received IFNAR1 neutralizing
antibody. 24 h later splenocytes were harvested, ACK-treated, and stained for flow cytometry as in
Methods. The MFI is displayed. DCs, monocytes, neutrophils, NK cells, and macrophages were gated as
in Fig. 7B. n=4. Data (representative of 2 independent experiments) were analyzed by one-way ANOVA
with Tukey’s correction for multiple comparisons. *P<0.05; **P<0.01; ***P < 0.001; ****P<0.0001.
CD11b+ DCs
PBSCDN
CDN + a
nti IF
NAR10
1000
2000
3000
4000
IL-1
5RA
MF
I
**** *****
CD11b- DCs
PBSCDN
CDN + a
nti IF
NAR10
1000
2000
3000
4000
IL-1
5RA
MF
I
**** ****ns
Macrophages
PBSCDN
CDN + a
nti I
FNAR10
2000
4000
6000
IL-1
5RA
MF
I
**** *******
Monocytes
PBSCDN
CDN + a
nti IF
NAR10
2000
4000
6000
8000
IL-1
5RA
MF
I
**** *****
Neutrophils
PBSCDN
CDN + a
nti IF
NAR10
2000
4000
IL-1
5RA
MF
I
**** *******
NK cells
PBSCDN
CDN + a
nti IF
NAR10
1000
2000
3000
IL-1
5RA
MF
I
**** ****
Spleen IL-15RA Expression
Fig. S14. Representative flow plots for Fig. 7C.
PB
SC
DN
CD
N +
IL
-15/
15R
ab
%IFN- %CD107a+ GzmB MFI %Sca-1+
10.1%
31.3%
20.0%
11.0%
41.4%
28.2%
904
2,442
1,633
11.6%
82.2%
GzmB
NK
1.1 70.4%
Sca1
NK
1.1
CD107a
NK
1.1
IFN-
NK
1.1
Tumor-draining LN NK cells
103
103
103
103
103 103
103
103 103 1041050
103
104
105
0
103
103 1040
103
104
105
0
1040
103
104
105
0
1041050
103
104
105
0
104 1050
103
104
105
0
104 1050
103
104
105
0
104 1050
103
104
105
0
104 1050
103
104
105
0
104 1050
103
104
105
0
104 1050
103
104
105
0
104 1050
103
104
105
0
103104 1050
103
104
105
0
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