caraerizzazionedicelluleimmunosoppressorie ... 14 novembre/castella.ppt.pdf ·...
TRANSCRIPT
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
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
MGUS
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
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
γδ T cell γδ T cell
In vitro inhibi5on of MDSC ac5vity: IDO inhibitor
MDSC γδ T cell
IDO Prolifera5on X 1-‐me5ltriptophan
γδ T cell γδ T cell
γδ T cell γδ T cell
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
°
* °
Inhibitory pathways in tumor microenvironment
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
°
* °
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+
Inhibitory pathways in tumor microenvironment
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
osteoblasts Stromal cells osteoclast
γδ T cell
MDSC
Plasmacells
PD-‐1
PDL-‐1
PDL-‐1 MDSC MDSC
PD-‐1
The importance of PD-‐1/PD-‐L1 axis in BM microenvironment
PI3K
Akt
Ac5va5on
Prolifera5on
CKs produc5on
osteoblasts Stromal cells osteoclast
γδ T cell
Plasmacells
PD-‐1
PDL-‐1
PD-‐1
The importance of PD-‐1/PD-‐L1 axis in BM microenvironment
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