Caraerizzazionedicelluleimmunosoppressorie ... 14 NOVEMBRE/Castella.ppt.pdf ·...

Barbara Castella Laboratorio di Ematologia Oncologica, CeRMS

Cara+erizzazione di cellule immunosoppressorie nel microambiente tumorale del mieloma mul5plo

4° WORKSHOP NAZIONALE della Società Italiana di Ematologia Sperimentale 14-‐15 Novembre 2013

Adapted from “Mul<ple myeloma: evolving gene<c events and host interac<ons”. Kuehl WM, Bergsagel PL Nat Rev Cancer 2002; 2(3):175-‐87

MM disease: gene5c altera5ons and microenvironment perturba5on

Immune dysregula5on

myeloma cell

MM immune microenvironment: from surveillance to escape

Treg

Foxp3

CD25

CD4

CD127

MDSC CD33 IL-‐4Rα

CD11b CD15

NKg2D

CD56

CD16

NK

αβ T cell

mDC iDC

Th17 cell

Adapted from “Immunologic microenvironment and personalized treatment in mul<ple myeloma”, Rossi M et al Expert Opin. Biol. Ther. (2013) 13:S83-‐S93

myeloma cell


Treg

Foxp3

CD25

CD4

CD127


CD11b CD15

NKg2D

CD56

CD16

NK

αβ T cell

mDC iDC

Th17 cell


MGUS

myeloma cell


Treg

Foxp3

CD25

CD4

CD127


CD11b CD15

NKg2D

CD56

CD16

NK

αβ T cell

mDC iDC

Th17 cell


MGUS MM

“Myeloid-‐derived suppressor cells as regulators of the immune system”, Gabrilovich DI, Nagaraj S, Nat Rev Immunol, 2009 Mar;9(3):162-‐74.

-‐  heterogeneous populaSon of cells of myeloid origin -‐  potent suppressors of various T-‐cell funcSons -‐  human phenotype: Lin-‐HLA-‐DRlow/-‐CD33+ or CD11b+CD14-‐CD33+; CD15+ in PB -‐  accumula5on in lymphoid organs and in tumors -‐  differenSaSon from tumor-‐associated macrophages (TAMs) in tumor Sssues

Myeloid-‐derived suppressor cells (MDSC): origin and defini5on

MDSC in MM pa5ents: what is known?

Mul%ple myeloma induces the immunosuppressive capacity of dis%nct myeloid-‐derived suppressor cell subpopula%ons in the bone marrow. Van Valckenborgh E, Schouppe E, Movahedi K, De Bruyne E, Menu E, De Baetselier P, Vanderkerken K, Van Ginderachter JA. Leukemia. 2012 Nov;26(11):2424-‐8. Myeloid-‐derived suppressor cells regulate growth of mul%ple myeloma by inhibi%ng T cells in bone marrow. Ramachandran IR, Martner A, Pisklakova A, Condamine T, Chase T, Vogl T, Roth J, Gabrilovich D, Nefedova Y. J Immunol. 2013 Apr 1;190(7):3815-‐23. Tumor-‐promo%ng immune-‐suppressive myeloid-‐derived suppressor cells in the mul%ple myeloma microenvironment in humans. Görgün GT, Whitehill G, Anderson JL, Hideshima T, Maguire C, Laubach J, Raje N, Munshi NC, Richardson PG, Anderson KC. Blood. 2013 Apr 11;121(15):2975-‐87

Ramachandran I.R. et al., JI 2013, 190:3815-‐3823

Increased frequency of MDSC in PB and BM of MM pa5ents

CD11b+CD14-‐CD33+ CD11b+CD14-‐CD33+CD15+ CD11b+CD14-‐CD33+CD15-‐

% M

DSC po

si5v

e cells

CTRL (n=19)

MM (n=47)

CTRL (n=10)

MM (n=100)

PB BM

0

20

40

60

80 % CD33+CD11b+CD14-‐

% CD11b+CD15+HLA-‐DRlow

°

° * p<0,001 ° p<0,001 ^ p<0,001

* ^

^

*

Increased frequency of MDSC in PB and BM of MM pa5ents CD

11b

CD33

Backgated on CD14-‐

PB BM

CTRL

MM 65%

8% 5%

38%

Inhibiton of conven5onal αβ T-‐cell prolifera5on by MM MDSC

Ramachandran I.R. et al., JI 2013, 190:3815-‐3823

MDSC (BM MM)

T(OKT3+IL2) (PB CTRL)

MDSC (BM MM) + T(OKT3+IL2) (PB CTRL)

0

5000

10000

15000

20000

Cpm H

3 TdR

Mechanisms of MDSC suppressive ac5vity

MDSC

killing

cytokine producSon

DC

Arginase TGFbeta IL-‐10

M2 polarizaSon?

maturaSon

migraSon

T cell sSmulaSon

NKg2D

CD56 CD16

NK

CD68 CD14

CD206

TAMs

αβ T cell

Arginase

iNOS ROS

proliferaSon

cytokine producSon

cytotoxicity

IL10

Foxp3

CD25

CD4

CD127

Tregs

proliferaSon

CD16

NKg2D

LFA1

γδ T cell

TCR

Adapted from “Myeloid-‐derived suppressor cells: linking inflamma<on and cancer”, Ostrand-‐Rosenberg, Journal of immunology, 2009

NKG2D

KIR LFA-1

TCR

Cytokines: IFN-g TNF-a

Chemokines: MIP-1a/b RANTES

Cytotoxic activity: Perforin Granzyme

CD16

ADCC

Vγ9Vδ2 T cell

Apo-A1 F1-ATPase

Mev pathway

IPP

MIC A/B ULBPs

ICAM-1

MHC-I ILT-2

tumor cell

Major determinants of intrinsic cancer cell susceptibiliy

1.  spontaneous IPP generation

2.  abundance of KIR ligands

3.  paucity of KAR ligands

4.  lack of MHC class I molecules

5.  costimulatory molecules

NHL/CLL

CML

solid cancers

non-human cancers

Vg9V

d2 T

-cel

l sus

cept

ibili

ty

NHL/MM high

low

absent

Castella B. et al, Cell Mol Life Sci. 2011; 68(14):2419-32

N130 PB IL2 gd/3.001

100 101 102 103 104gammadelta FITC

3.6%

TCR TCR gd

CD3

Vγ9Vδ2 T cells in the peripheral blood: a rare subpopula5on

NKG2D

KIR LFA-‐1

TCR

Vγ9Vδ2 T cell

Apo-‐A1 F1-‐ATPase

Mev pathway

MIC A/B ULBPs

ICAM-‐1

MHC-‐I ILT-‐2

APC

IPP

Zoledronate

Tumor cell

prolifera5on

Vγ9Vδ2

γδ TCR

CD3

R

MM

Mariani , et al. Leukemia 2005; 19:664-‐70

NR

Approx 50% of MM pa5ents at diagnosis are refractory to ZA-‐induced monocyte-‐dependent IPP s5mula5on

Castella, et al. J Immunol. 2011; 187:1578-‐90

65 %

DCZA+

8 %

PBMCZA+

MM

CD3

γδ TCR

but DC ZA-‐treated allow to recover the prolifera5on of NR pa5ents

Impaired pAg-‐induced prolifera5on in BM Vγ9Vδ2 T cells of MM: BM-‐derived DCs fail to recover Vγ9Vδ2 T-‐cell prolifera5on

PBMC (n=11)

DC(PB) +PBL (n=11)

0

1e+5

3e+5

2e+5

PB

Viab

le γδ T cells/w

ell

IL2 ZA+IL2

*p=0,04

*

*

PBMC (n=35)

BM

0

1e+5

2e+5

3e+5

BMMC (n=18)

DC(BM) +BML (n=18)

IL2 ZA+IL2

BMMC (n=58)

Viab

le γδ T cells/w

ell

0

20000

40000

60000

80000 IL2 ZA+IL2

NKg2D

CD56

CD16

NK

Monocytes

CD16 CD14

γδ T cell

TCR

Plasmacells

Tregs

Foxp3 CD25

CD4

CD127

CD11b

CD15

CD33

MDSC

IL-4Rα

CD68 CD14

CD206

TAMs

Bone marrow: MM tumor bed

Monocytes

CD16 CD14

CD16

NKg2D

LFA1

γδ T cell

αβ T cell

DC

T EM

T EM T EM DC

NKg2D

CD56

CD16

NK

osteoblasts

Stromal cells

osteoclast

osteoblasts

Stromal cells

osteoclast

αβ T cell

DC

T EM

T EM T EM

NKg2D

CD56

CD16

NK

Monocytes

CD16 CD14

CD16

NKg2D

LFA1

γδ T cell

TCR

Plasmacells

Inhibitory cells in the bone marrow: MDSC

Tregs

Foxp3 CD25

CD4

CD127

CD11b

CD15

CD33

MDSC

IL-4Rα

CD68 CD14

CD206

TAMs

DC

Wesolowski et al. J ImmunoTherapy of Cancer 2013

Deac5va5on of MDSC

Differen5a5on of MDSC into mature cells

Blocking development of MDSC

Deple5on of MDSC

Vitamins (ATRA, Vitamin A) Cytokynes (IL-‐12)

Bisphoshponates (Zoledronic acid)

Modulators of cell signalling (JAK2/STAT3; VEGF inhibitors)

NO inhibitors (PDE5 inhibitors, L-‐NAME)

Arginase inhibitors (COX2 inhibitors, NOHA) ROS inhibitors

MDSC migra5on inhibitors (anS-‐glican anSbodies)

Strategies of MDSC inhibi5on under inves5ga5on

Cytotoxic agents (gentamicine, cisplaSn)

HSP90 inhibitors IL-‐6 blockers

IL-‐2 ZA

Sildenafil

+ - -

+ + -

+ + +

BMMC culture (n=10)

0

5000

10000

15000

20000

Viab

le γδ T cells/w

ell

MDSC γδ T cell

PDE5

In vitro inhibi5on of MDSC ac5vity: PDE5 inhibitor

Prolifera5on X sildenafil

γδ T cell γδ T cell


In vitro inhibi5on of MDSC ac5vity: IDO inhibitor

MDSC γδ T cell

IDO Prolifera5on X 1-‐me5ltriptophan



0

5000

10000

15000

20000

IL-‐2 ZA

1-‐MT

+ - -

+ + -

+ + +

Viab

le γδ T cells/w

ell BMMC culture (n=6)

MDSC in vitro deple5on

BMMC

0

5000

10000

15000

20000

IL-‐2

ZA

+

-‐

+

+

Viab

le Vγ9Vδ

2 T cells/w

ell

BM COSTANZO 33/14/11b.001

0 200 400 600 800 1000Forward Scatter

R1

BM COSTANZO 33/14/11b.001

100 101 102 103 104CD33 FITC

78%

BMMC

CD33

CD11b

MDSC-‐depleted BMMC

+

+

+

-‐

MDSC- 33/14/3/11b.001

0 200 400 600 800 1000Forward Scatter

R1

MDSC- 33/14/3/11b.001

100 101 102 103 104CD33 FITC

CD33

CD11b

6%

MDSC-‐depleted BMMC

Tumor cell

MDSC Vγ9δ2 T cell

Inhibitory pathways in tumor microenvironment

MM CTRL

BM (n=64)

PB (n=35)

BM (n=5)

PB (n=12)

% Vγ9Vδ

2+ PD-‐1+ T cells

0

5

10

15

20

*

* p< 0.001

° p=0.004

°

* °


Tumor cell

MDSC Vγ9δ2 T cell


MM CTRL

BM (n=64)

PB (n=35)

BM (n=5)

PB (n=12)

% Vγ9Vδ

2+ PD-‐1+ T cells

0

5

10

15

20

*

* p< 0.001

° p=0.004

°

* °

MM

BM (n=24)

PB (n=12)

BM (n=2)

PB (n=4)

CTRL

% M

DSC posi5ve cells 0

20

40

60

BM MM (n=15)

% CD1

38+ po

si5v

e cells

0

20

40

60

80

PD-‐L1+ PD-‐L2+


IL-‐2 ZA

α-‐PD-‐1 (μg/ml)

+ -‐ -‐

+ + -‐

+ + +

(0.1)

+ + + (1)

+ + +

(30)

0

20000

40000

60000

Viab

le Vγ9Vδ

2 T cells/w

ell

0

10

20

30

40

50

60

70

IL-‐2 ZA α-‐PD-‐1

+ -‐ -‐

+ + -‐

+ -‐ +

+ + +

% Vγ9Vδ2 + CD107+

p < 0.05

Par5al recovery of BM Vγ9Vδ2 T cells reac5vity upon PD-‐1 blockade

Clinical trial with blocking mAb: -‐  CT-‐011: bind to human PD1

-‐  MDX-‐1106: blocks binding of PD1 to PDL1 and PDL2

Ac5va5on

Survival

Cytokine produc5on Prolifera5on

osteoblasts Stromal cells osteoclast

γδ T cell

MDSC

Plasmacells

MDSC MDSC

The importance of PD-‐1/PD-‐L1 axis in BM microenvironment


γδ T cell

MDSC

Plasmacells

PD-‐1

PDL-‐1

PDL-‐1 MDSC MDSC

PD-‐1


PI3K

Akt

Ac5va5on

Prolifera5on

CKs produc5on


γδ T cell

Plasmacells

PD-‐1

PDL-‐1

PD-‐1


PI3K

Akt

Ac5va5on

Prolifera5on

CKs produc5on

MDSC

PDL-‐1 MDSC MDSC

Chemotherapy

Remission

DIA (N=121)

REM (N=44)

REC (N=18)

0

20

40

60

80

% M

DSC po

si5v

e cells

p < 0,0001 p = 0,005

Frequency and PD-‐L1 expression in BM MDSC of MM pa5ents according to disease status

DIA (N=15)

REM (N=9)

REC (N=2)

0

20

40

60

% PDL

1 on

MDS

C po

si5v

e cells

0

20

40

60

80

100

% Vgamma9+ PD

1+

DIA (n=65)

REM (n=13)

REC (n=7)

PD-‐1 expression on MM BM Vγ9Vδ2 T cells and pAgs-‐reac5vity according to disease status

DIA (n=56)

REM (n=12)

REC (n=4)

0

20000

40000

60000 ZA-‐

ZA+

Viab

le Vγ9Vδ

2 T cells/w

ell

Conclusions

MDSC are increased in the BM of MM pa5ents at diagnosis and persist in high numbers aser induc5on of clinical remission PD-‐L1 expression on MDSC could represent an addi5onal suppressive mechanism in MM BM microenvironment The PD-‐1/PD-‐L1 axis is a poten5al target for the immunotherapy of mul5ple myeloma, even in complete remission aser chemotherapy

Hematology-‐Oncology Laboratory, CERMs Torino, Italy

Thanks to all!

Prof. Massaia Massimo

Patrizia Sciancalepore

Myriam Foglie+a

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