Supplementary Figure 1 *** a WT.VK Myc *** WT.VK Myc *** ** c...
Transcript of Supplementary Figure 1 *** a WT.VK Myc *** WT.VK Myc *** ** c...
0.2
0.2
20
25
95
98
3.9
1.9
B220
CD
138
WT
Cd226-/-
VK*Myc+ VK*Myc-
CD155 B220
CD
138
CD155
Supplementary Figure 1
d
Supplementary Figure 1. a,b. C57BL/6 Cd226+/+ (n=55), Cd226–/+ (n=27) and Cd226–/– (n=25) Vk*MYC transgenic mice and Cd226+/+ (n=12), Cd226–/+ (n=15) and Cd226–/– (n=9) non-‐transgenic liIermate controls were monitored for MM development (a) and survival (b). a. The absence of CD226 is not impacSng spontaneous MM emergence in Vk*MYC transgenic mice. Graph showing the percentage of the indicated strains of mice with serum paraproteinemia over Sme. b. Histogram showing a significant decrease in the survival of Cd226–/– Vk*MYC mice as compared to WT and Cd226–/+ Vk*MYC liIermates. *p<0.05, ***p<0.001, Mann Whitney test. c. Graph showing the percentage of CD138+B220-‐ PCs analysed by flow cytometry in the BM of the indicated group of mice at necropsy. d. The expansion of plasma cells CD138+B220-‐ expressing CD155+ is mainly restricted to the BM in Vk*MYC mice. RepresentaSve FACS plots showing the percentage of CD138+B220-‐ PCs and their level of CD155 expression in the bone marrow of Cd226+/+ (n=15) and Cd226–/– (n=14) Vk*MYC transgenic mice and in Cd226+/+ (n=6) and Cd226–/– (n=10) non-‐transgenic liIermate controls at 450 days of age. e. RepresentaSve FACS plot showing the frequency of CD138+CD155+ malignant plasma cells in the spleen of the same strains of mice at 450 days of age.
CD
138
CD155
0.1 0.1 0.2 0.4
e VK*Myc+ VK*Myc-
WT Cd226-/- WT Cd226-/-
200
400
600
800
1000
Surv
ival
(day
s)
******
* DNAM-I -/-.VK Myc +
DNAM-I +/-.VK Myc +
WT.VK Myc +
DNAM-I -/-
DNAM-I +/-
WT
200
400
600
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1000Su
rviv
al (d
ays)
******
* DNAM-I -/-.VK Myc +
DNAM-I +/-.VK Myc +
WT.VK Myc +
DNAM-I -/-
DNAM-I +/-
WTCd226+/+
Cd226–/–
Cd226–/–.VK*Myc+
Cd226+/–.VK*Myc+
Cd226+/+.VK*Myc+
200
400
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1000Su
rviv
al (d
ays)
******
* DNAM-I -/-.VK Myc +
DNAM-I +/-.VK Myc +
WT.VK Myc +
DNAM-I -/-
DNAM-I +/-
WT
200
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1000
Surv
ival
(day
s)
******
* DNAM-I -/-.VK Myc +
DNAM-I +/-.VK Myc +
WT.VK Myc +
DNAM-I -/-
DNAM-I +/-
WTCd226+/–
0 200 400 600 8000
20
40
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age of mice
Inci
denc
e of
M-s
pike
s (%
)
WT.VK Myc +DNAM-I +/-.VK Myc +
DNAM-I -/-.VK Myc +WT DNAM-I -/-
DNAM-I +/-
a
200 400 600 8000
20
40
60
80
100
Days
Perce
nt surv
ival
DNAM-I -/-.VK Myc +DNAM-I +/-.VK Myc +
WT.VK Myc +
WT
DNAM-I -/-DNAM-I +/- ** ***
200 400 600 8000
20
40
60
80
100
Days
Perc
ent s
urvi
val
DNAM-I -/-.VK Myc +DNAM-I +/-.VK Myc +
WT.VK Myc +
WT
DNAM-I -/-DNAM-I +/- ** ***Cd226+/+ Cd226+/– Cd226–/–
Cd226–/–.VK*Myc+
Cd226+/–.VK*Myc+
Cd226+/+.VK*Myc+
b
Cd226–/–
Cd226+/–
Cd226+/+
Cd226–/– .VK*Myc+
Cd226+/+ .VK*Myc+
Cd226+/– .VK*Myc+
0
10
20
30
40
BM p
lasm
a ce
lls (%
)
c
a
Supplementary Figure 2 : Transplantable VKMYC model. a-‐b. 2 x 106 Vk12653 cells were injected i.v into 10 Rag2–/–ilr2g–/– mice. a. RepresentaSve FACS plot showing the percentages and the phenotype of malignant plasma cells in the spleen from Rag2–/–ilr2g–/– mice 5 weeks aeer injecSon. b. RepresentaSve FACS plot showing the expression of the indicated markers (open line) on VK12653 CD138+B220-‐ plasma cells as compared to isotype control staining (filled line). c. RepresentaSve m-‐spikes obtained with the Vk12653 and Vk12598 cell lines d. Table showing the total expression value of the indicated genes analyzed by RNA sequencing in CD138+B220-‐ plasma cells purified from 3 different MM bearing VK*MYC transgenic mice (VKMYC1-‐3) or VK12598 MM cells. e. RepresentaSve FACS plot and graphs showing the percentages of malignant plasma cells in the spleen and the BM of WT mice 2 and 6 weeks aeer Vk12653 MM cell injecSon.
Spleen
Naive
CD138
CD155
0.00 0.03
0.19 0.00
Week 2
Bone marrow
39.8
42.7
Week 5
BM
PC
s (%
)
Spl
een
PC
s (%
)
d
B220
CD
138
CD155 CD3
NK
1.1 58 59
e
Gene alias chr start stop strand VkMYC 1 VkMYC 2 VkMYC 3 Vk12598
Pvr CD155 7 20488927 20506493 - 7.98 9.43 9.37 6.57 Pvrl2 CD112 7 20301993 20334923 - 2.35 0.06 1.32 1.73 Tnfsf9 CD137L 17 57244808 57247181 + 0.14 0.71 0.07 0.56 Tnfrsf9 CD137 4 150294264 150320212 + 0.05 0.32 0.00 0.05 Ctla4 CTLA-4 1 60965869 60972675 + 0.11 6.44 0.20 0.00 Cd226 DNAM1 18 89366819 89439720 + 0.06 0.01 0.00 0.00 Pdcd1 PD-1 1 95934882 95949131 - 0.07 0.27 0.02 0.42 Pdcd1lg2 PD-L2 19 29485409 29547418 + 0.14 0.66 0.05 0.00 Cd274 PD-L1 19 29441928 29462585 + 7.59 18.59 22.99 8.21
!e
b
CD155 Rae-1 H2Db H2Kb
CD137 CD137L PD-L1 PD-L2 CTLA-4
CD112
Supplementary Figure 2
Albumin α1 α2 β1 β2
Vk12598
Vk12653
M-‐Spike
c
16.7 46.6
CD155
CD
138
WT Cd226-/-
Supplementary Figure 3
a
13.2 39.7
CD155
CD
138
WT Cd226-/-
c
WT
DNAM-1-/-
WT
DNAM-1-/-
WT
DNAM-1-/-
0
20
40
60
80
MM
in B
one
Mar
row
(%)
Naive 1 x 106 MM 2 x 106 MM
***
WTDNAM-1-/-
WTDNAM-1-/-
WTLegend
WT
DNAM-1-/-
WT
DNAM-1-/-
WT
DNAM-1-/-
0.0
5.0×105
1.0×106
1.5×106
2.0×106
2.5×106
Num
ber o
f MM
cel
ls /
Bon
e M
arro
w
Naive1 x 106 MM2 x 106 MM
**
BM
PC
s (%
)
Num
ber o
f BM
PC
s
WT
DNAM-1-/-
WT
DNAM-1-/-
WT
DNAM-1-/-
0
20
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80
MM
in B
one
Mar
row
(%)
Naive 1 x 106 MM 2 x 106 MM
***
WTDNAM-1-/-
WTDNAM-1-/-
WTLegend
WT Cd226-/- WT Cd226-/- WT Cd226-/-
No MM
1x106 MM cells
2x106 MM cells
b
Supplementary Figure 3 : CD226 and tumor CD155 is required for the immune-‐control of MM. a-‐c. WT or Cd226–/– mice were challenged i.v with 2x106 (a, b, c) or 1x106 (c) Vk12653 MM cells and the number and the percentage of malignant PCs was determined by flow cytometry in the spleen and in the BM 5 weeks aeer injecSon. a. RepresentaSve FACS plots showing the frequency of CD138+ CD155+ malignant PCs in the bone marrow (bo-om) and the spleen (top) of the indicated strains of mice. b. Spleen weight of the indicated groups of mice as in a. RepresentaSve experiment out of 4 involving groups of n=10 mice. c. Graphs showing the mean percentage and number of B220–CD138+ PCs in the bone marrow of the indicated strains of mice. Each symbol represents one individual mouse. d. WT or Cd226–/– mice were challenged i.v with or 1x106 Vk12653 MM cells and the survival was monitored. RepresentaSve experiment involving groups of n=10 mice. *p<0.05, **p<0.01, ***p<0.001. Mann Whitney test (b, c) and Mantel Cox test (d) .
Wild TypeDNAM-1-/-Wild TypeDNAM-1-/-
0.0
0.5
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1.5
2.0
2.5 ***
Sple
ens'
wei
ght (
gram
s)
● ○
Wild TypeDNAM-1-/-
WT Cd226-/-
No MM
WT
Cd226-/- 2x106 MM cells
Spl
een
wei
ght (
g)
d S
urvi
val (
%)
Time (days)
WT Cd226-/- **
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50
100
Time
Perc
ent s
urvi
val
WTDNAM KO **
0 30 40 50 600
50
100
Time
Perc
ent s
urvi
val
WTDNAM KO
Supplementary Figure 4
Supplementary Figure 4: NK cells and CD8+ T cells have non-‐redundant anO-‐myeloma acOvity through perforin and IFN-‐γ pathways. a-‐b. WT mice were injected with control IgG, anS-‐AsGM1 and/or anS-‐CD8 to deplete NK cells and/or CD8+ T cells, and were challenged with Vk12653 cells. The spleen weight (a) and the number of PCs in the spleen (b) were measured 5 weeks aeer injecSon. RepresentaSve experiments out of 2 involving groups of n=10 mice. Each symbol represents one individual mouse. c. WT, Pfp–/– and Ifng–/– mice were injected i.v with 2 x 106 Vk12653 cells. The spleen weight was measured 5 weeks aeer injecSon. RepresentaSve experiments out of 2 involving groups of n=10 mice. Each symbol represents one individual mouse. ns p>0.05, *p<0.05, **p<0.01, ***p<0.001. Mann Whitney test.
WT
PFP-/-
IFNy-/-
0
1
2
3
4
Sple
en w
eigh
t (g) ns
**a
Contro
l IgG
anti-a
GM1
anti-C
D8
anti-a
sGM1+
anti-C
D8105
106
107
108
109
1010
Num
ber o
f SP
L pl
asm
a ce
lls ******
Control IgG anti-aGM1anti-CD8anti-asGM1+anti-CD8
b
Contro
l IgG
anti-a
GM1
anti-C
D8
anti-a
sGM1+
anti-C
D8105
106
107
108
109
1010
Num
ber o
f SP
L pl
asm
a ce
lls ******
Control IgG anti-aGM1anti-CD8anti-asGM1+anti-CD8
cIg anti-AsGM1
anti-AsGM1+anti-CD8 anti-CD8 c
0
1
2
3
4
Sple
en w
eigh
t (g)
IgG aGM1CD8aGM1+CD8
***
**0
1
2
3
4
Sple
en w
eigh
t (g)
IgG aGM1CD8aGM1+CD8
***
**
cIg anti-AsGM1
anti-AsGM1+anti-CD8 anti-CD8
WT Pfp–/– Ifng-/-
Supplementary Figure 5
Supplementary Figure 5 : OpOmal anO-‐myeloma therapy requires CD226. (a-‐c) WT and Cd226–/– mice were injected i.v with 2 x 106 Vk12653 MM cells. Aeer 3 weeks, mice were injected either with cIg or anS-‐CD226 mAbs and were subsequently treated with PBS, Btz (i.p; 0.5 mg/kg) or CTX (i.p; 50 mg/kg) twice a week. a. γ-‐globulin levels in the serum of WT and Cd226–/– mice before therapy 3 weeks aeer MM injecSon. b-‐c. graphs showing the percentages and the numbers of CD138+CD155+ PCs in the spleen (b) and in the BM (c) of the indicated groups of mice at the end of the treatment. Data are pooled from two experiments. Each symbol represents one individual mouse. d. WT and Cd226–/– mice were injected i.v with 2 x 106 Vk12653 MM cells. Aeer 3 weeks, mice were treated with PBS or CTX (i.p; 20 mg/kg) twice a week and the survival of the indicated groups of mice was monitored. Experiment involving groups of n=10 mice. *p<0.05, **p<0.01. Mann Whitney test. The vehicle groups displayed in part c have been presented in Figure 6H and are derived from the same set of experiments.
WT
DNAM-1- /-
0
2
4
6
% o
f M-S
pike
on
wee
k 3 *
a Serum γ-‐globu
lin (%
)
WT Cd226–/– 0
20
40
60
80 ****
***
** *******
0
20
40
60
80
******
*** ******** DNAM-1–/– Vehicle
Cyclophosphamide
IgG BtzWT + α-CD226 Cd226–/–
WT + cIg
Veh CTX Btz
Spleen
PC (%
)
BM PC (%
)
DNAM-1–/– Vehicle
Cyclophosphamide
IgG BtzWT + α-CD226 Cd226–/–
WT + cIg
Veh CTX Btz
b
c
Num
ber o
f spl
een
PC
s
104105106107108109
*****
***
*** *****
Veh CTX Btz
0 30 40 50 60 70 800
50
100
Days
Perc
ent s
urvi
val
**DNAM KO PBSDNAM KO CycloWT PBSWT Cyclo
0 30 40 50 60 70 800
50
100
Days
Perc
ent s
urvi
val
**DNAM KO PBSDNAM KO CycloWT PBSWT Cyclo
Cd226–/–
WT Cd226–/– + CTX
WT + CTX **
n.s d
Supplementary Figure 6. TherapeuOc efficacy of anO-‐CD137 immunotherapy against myeloma. (a-‐b) WT mice were injected with control Ig, anS-‐CD137, anS -‐PD-‐1 or anS-‐CTLA-‐4 as described in the Material and Methods and subsequently challenged with 2 x 106 Vk12653 MM cells. RepresentaSve FACS plot showing the percentages (a) and the numbers (b) of malignant CD138+CD155+ PCs in the spleen of the indicated groups of mice at the end of the treatment. Data are representaSve of two independent experiments involving groups of n=10 mice. Each symbol represents one individual mouse. c-‐d WT or Cd226–/– mice were injected with cIg or anS-‐CD137 and then challenged with 2 x 106 Vk12653 MM cells. c,d. RepresentaSve FACS plot and graph showing the percentages (c), the mean numbers (d) + SEM of malignant CD138+CD155+ PCs in the spleen and in the BM of the indicated groups of mice at the end of the treatment. Data are representaSve of two independent experiments involving groups of n=5 mice. *p<0.05, **p<0.01, ***p<0.001. Mann Whitney test.
a
Supplementary Figure 6
b
Num
ber o
f SP
L P
Cs
cIg α-‐CD137 α-‐CTLA-‐4 α-‐PD-‐1
WT C
ontrol Ig
G
WT A
nti-CD13
7
WT A
nti-CDTLA4
WT A
nti-PD1
104105106107108
Num
ber
of M
M o
n S
PL
**WT Control IgGWT Anti-CD137WT Anti-CDTLA4WT Anti-PD1
31.3 0.9 32.3 33.6 CD
138
CD155
WT IgG
WT aCd13
7
DNAM-1-/- Ig
DNAM-1-/- Anti-C
D137
104105106107108109
**
***
**
WT IgG
WT aCd13
7
DNAM-1-/- Ig
DNAM-1-/- Anti-C
D137
103
104
105
106
107 ** *
Num
ber o
f BM
PC
s N
umbe
r of s
plee
n P
Cs
WT IgG
WT aCd13
7
DNAM-1-/- Ig
DNAM-1-/- Anti-C
D137
0
20
40
60
80
BM p
lasm
a ce
lls (%
)
****
WT IgG
WT aCd13
7
DNAM-1-/- Ig
DNAM-1-/- Anti-C
D137
0
20
40
60
80 **
**
****
Spl
een
PC
s (%
) B
M P
Cs
(%)
49.2 0.08
75.6 0.08
14.7 0.2
62.8 14.5
CD13
8
CD155
Cd226–/–
WT
Cd226–/–
WT
WT IgG WT aCd137DNAM-1-/- IgDNAM-1-/- Anti-CD137 Cd226–/– cIg
WT cIg
α-‐CD137 cIg
Spleen
BM
Cd226–/– α-‐CD137
WT α-‐CD137
c d
Ig α-‐CD137 α-‐CTLA4 α-‐PD1
0
10
20
30
40
50
% o
f P
D1+
cells
0
20
40
60
80
% o
f C
TLA
4+ ce
lls
Vk*mycNaive
CD
137+
cel
ls (%
)
PD
-1+
cells
(%)
CTL
A-4
+ ce
lls (%
)
0
10
20
30
40
50%
of
CD
137+
cells
0
20
40
60
80
100%
of
PD
1+ ce
lls
PD1 BM week6
***
******
0
20
40
60
80
CD137 BM week6
% o
f C
D13
7+ ce
lls *** *** ***
CD
137+
cel
ls (%
)
PD
-1+
cells
(%)
CTL
A-4
+ ce
lls (%
) b
a
CD8/Tregs raS
o
20
40
60
0
Tregs a
mon
g CD
4+ T
cells (%
)
CD8 am
ong lymph
ocytes (%
)
4
8
12
0 2
6
10
20
0 10
30 40
Supplementary Figure 7. Mechanism of anO-‐CD137 immunotherapy against myeloma. (a-‐b) WT mice were injected with with 2 x 106 Vk12653 MM cells. Graphs are showing the percentages of CD137+, PD-‐1+ or CTLA-‐4+ cells among the indicated populaSons of lymphocytes within the BM of WT mice 2 weeks (a) or 6 weeks aeer MM cells injecSon (b). Data are representaSve of two independent experiments involving groups of n=5-‐10 mice. Each symbol represents one individual mouse. c-‐d. Mice were injected i.v with 2 x 106 Vk12653 MM cells. Aeer 3 weeks, mice with established MM were injected either with IgG, anS-‐CD137 or anS-‐PD-‐1 mAbs. c. Graphs are showing the percentages + SEM of PD-‐1+ cells among the indicated populaSons of lymphocytes within the BM of WT mice 2 weeks aeer the beginning of the indicated treatment. Experiment involving groups of n=10 mice. d. Graphs are showing the percentage of the indicated populaSons of immune cells at the end of the indicated treatment. Data are pooled from two independent experiments. *p<0.05, **p<0.01, ***p<0.001. Mann Whitney test.
PD
-1+
NK
cel
ls (%
) c
PD
-1+
CD
8 ce
lls (%
)
PD
-1+
CD
4 ce
lls (%
)
PD
-1+
Treg
s (%
)
d
Supplementary Figure 7
Supplementary Figure 8. WT mice, CD226-‐/-‐ mice, IFN-‐γ-‐/-‐ mice and pfp-‐/-‐ mice were injected with 4x105 Vk12598 cells and survival was monitored over Sme. Data displayed here have already been shown in Figures 2E (WT and CD226-‐/-‐ mice) and 3F (WT and IFN-‐γ-‐/-‐ and pfp-‐/-‐ mice) and are from one experiment with n=9-‐12 mice per group. *** p<0.001; Mantel-‐Cox test.
Supplementary Figure 8
Supplementary Figure 9. WT mice and CD226-‐/-‐ were injected with 2x106 Vk12653 cells. Aeer 3 weeks, mice were treated with control IgG (CIg) or α-‐CD226 blocking anSbodies and subsequently treated with PBS (vehicle), CTX, Btz or α-‐CD137. Graphs show the % of serum γ-‐globulin (A), the numbers (B) and the % (C) of malignant CD138+CD155+ PCs in the BM of the indicated groups aeer 3 weeks of treatment. Data are pooled from 2 independent experiments. Data were already presented in Figures 5B and 6D (A), Figures 5D, 6E and 6H (B) and Figures 6H and Supp S5C (C). All the groups from the same set of experiments are shown together here. *P<0.05, **P<0.01, ***P<0.001; Mann-‐Whitney U test.
Supplementary Figure 9
A
B
C