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

800

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

600

800

1000Su

rviv

al (d

ays)

******

* DNAM-I -/-.VK Myc +

DNAM-I +/-.VK Myc +

WT.VK Myc +

DNAM-I -/-

DNAM-I +/-

WT

200

400

600

800

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

60

80

100

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

40

60

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

1.0

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-/- **  

0 20 40 60 80 1000

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.    

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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