LYMPHNODE&MESENCHYMAL&STROMAL&CELLS&(LNMSC)&& …

LYMPH NODE MESENCHYMAL STROMAL CELLS (LNMSC) REGULATE THE IMMUNE RESPONSE AGAINST LYMPHOMA

F C R S

DITID-‐ IRCCS and FCSR San Raffaele Division of Immunology

Transplants and Infec2ous Diseases Paolo Canevali

Caterina Camodeca Maria Raffaella Zocchi

UNIVERSITY of PISA DEPARTMENT of PHARMACY,

Elisa Nuti Armando Rossello Caterina Camodeca

IRCCS AOU San Mar2no-‐IST, UNIVERSITY of GENOA Unit of Molecular Oncology and Angiogenesis Department of Integrated Oncologic therapies Alessandra Musso Roberta Vené Alessandro Poggi Department of Pathology Annalisa Kunkl Jean-‐Louis Rave> Laboratory of Hematology and Clinical Oncohematology Silvia Catellani Ivana Pierri Marco Gobbi Department of Oncology, Biology and Gene2cs Sara Tavella

AIRC Compagnia di San Paolo

[email protected] [email protected]

Are cytotoxic T cells present in lymphomas? Infiltration of CD8+ GrzB+ cells in the interfollicular spaces of FL lymph nodes.

Laurent C et al. Blood 2011;118:5371-5379

©2011 by American Society of Hematology

Granzyme B is a prognostic marker of PFS in FL. The graph shows

progression-free survival (PFS) in the 80 patients scored by IHC after R-

combined chemotherapy.

Blood, prepublished online October 7, 2014; Arne Kolstad, Shraddha Kumari, Mateusz Walczak, Ulf Madsbu, Trond Hagtvedt, Trond Velde Bogsrud, Gunnar Kvalheim, Harald Holte, Ellen Aurlien, Jan Delabie, Anne Tierens and Johanna Olweus. Sequen2al intranodal immunotherapy induces an2-‐tumor immunity and correlated regression of disseminated follicular lymphoma Scheme of treatment: Low dose rituximab+single dose 8Gy radiotherapy+immature DC+GMCSF Key points: 1) Local immunotherapy induced systemic responses in pa\ents with disseminated follicular lymphoma 2) Clinical responses correlated with systemic an\-‐tumor T cell immunity. Cumula2ve survival Pa2ents with immune response 0.65 on 30th month Pa2ents without immune response 0.0 on 30th month

NKG2D-‐MICA,B, ULBP1-‐6 CD160 -‐HLAC

CD16(FcγRIIIa)-‐IgG CD94/NKG2C,E-‐HLAE

CD27-‐CD70 KIR2DS2,3,4,5,-‐HLA-‐C? DNAM1-‐PVR, nec2n2

KIR3DL3, KIR2DL5A, 5B-‐? KIR2DS1-‐HLA-‐C

CD224(SLAMF4, 2B4)-‐CD48 SLAMF7(CS1)-‐SLAMF7(CS1)

NKp30 (CD337)-‐B7H6, BAT3, CMVp65, HS

NKp80-‐AICL (CLE2B) CD96-‐NECL5

PSGL1-‐L-‐selec\n SLAMF6 (NTBA)-‐SLAMF6 (NTBA)

NKp44 (CD336 )-‐viral H6, NKp44L(MLL5), PCNA , HS

CD100-‐CD72 NKp46 (CD335 )-‐viral H6, HS

NCAM (CD56)-‐NCAM (CD56) α4β1 – FN, VCAM1

an2-‐tumor effector lymphocytes (NK, NKT, Tγδ, CTLs)

tumor

microenvironment associated

target cells as tumor B cells,

MSC TAF, CAF,myoF,

fibrocytes, pericytes T and non-‐T cells MDSC, APC,

endothelial cells Treg

LAIR1 (CD305)-‐Collagen I, III, IV, XII Siglec 3,7,9-‐ sialic acid

KLRG1-‐cadherin

NKRP1A-‐CLEC2D (LLT1)

CD94/NKG2A-‐HLAE LIR1 (ILT2, LILRB1)-‐HLA-‐I

KIR2DL1,2,3-‐HLA-‐C KIR3DL1,2,3-‐HLA-‐A or B

KIR3DL3, KIR2DL5A, 5B-‐?

If the inhibi\ng signal prevails: no target cell lysis reduced cytokines

produc\on

If the ac\va\ng signal prevails: target cell

lysis, cytokine produc\on

adhesion ↑ [Ca++]i ↑ kinases ↑

adhesion ↑ [Ca++]i ↑ kinases ↑

?

?

CEACAM1 (CD66a)-‐CEACAM1 (CD66a)

LFA1-‐ICAM1,2,3,4

PECAM1(CD31)-‐PECAM1(CD31)/CD38

NK,γδT, αβT cell

IFN-γ, TNF-α

Cytolysis ADCC

TCR/self-HLA-I+Ag (TAA) PD1/PDL1, CTLA-4/B7 family members NCR/B7 family members/viral products Natural Cytotoxicity Receptors (NKp30, NKp44, NKp46) NKG2D/NKG2DL as ULBP-MICA/MICB Natural Killer group 2D protein interacts with UL16 binding protein or MHC-related protein A IRS/self-HLA-I Inhibitory Superfamily Receptors (KIR, CLIR, ILT) missing self hypothesis Activating isoforms of IRS? LAIR1/collagen 2B4/CD48 DNAM1/PVR-nectin family members

Anti-tumor effector cells Natural Killer, γδT, CD8+αβT lymphocytes

Lymph node pluripotent MSC CD90+CD105+CD73+

Sox2+ CD146+ CD40+ TGII+ CL+LN-‐

B7-‐1-‐B7-‐2-‐ B7-‐H1+ Erp5+, ADAM10/17+

MDSC

FDC

NK Tumor B

CTL B

Tumor B

Tumor B

Tumor B

Tumor B

Tumor B

Tumor B

FDC

MSC

MSC

Lymphoma microenvironment

HL and NHL NKG2D-‐NKG2DL Erp5, ADAM10-‐17 NB-‐Ps γδ T cellls

γδ T cells

MSC

ERp5 is a disulfide isomerase Disulphide bonds exchange

Release of MICA/B-ERp5 complexes

ADAM10 is a disintegrin and metalloproteinase

MICA/B ULBP4-5 ULBP 1-3, 6 α1 α1 α1 α2

α2 α2α3

TM GPI TM

α1 α2 α3

sULBPs

sMICA/B

α1 α2 α1 α2

ERp5

ERp5

sNKG2DLs bind NKG2D

Inhibiting target cell recognition

X

Mesenchymal Stromal Cells

NKG2DL, ERp5 and ADAM10 expression in cHL.

C

0 1 2 3 4 5 6 7 8 9 10

mRN

A (ΔC T)

cHL Stromal areas (n=20) cHL Parenchyma(n=20)

B TG cHL NS CTR cHL NS

TG cHL MC CTR cHL MC

i ii

iii iv

0 2 4 6 8 10

mRN

A (ΔC T) Healthy LN

Ai cHL Nodular Sclerosing (NS, n=15)

A

0 1 2 3 4 5 6 7 8 9 10

mRN

A (ΔC T)

cHL Mixed Cellularity (MS, n=9)

Microdissec\on

TG staining= Stroma

TG staining= Stroma

0 1 2 3 4 5 6 7 8 9

10

mRN

A (ΔC T)

HL NS (n=14) HL MC (n=10) i

A HL S (n=10) HL P (n=10)

0 1 2 3 4 5 6 7 8 9

10

mRN

A (ΔC T)

ii

B

0

2

4

6

8

10 Healthy LN

mRN

A (ΔC T)

TGFβ transcription is higher in HL than in healthy LN and is prevalent in the stromal areas

MICRODISSECTION

stromal areas are identified by

transglutaminase

MIC-‐A ULBP3 A

ERp5 ADAM10

i ii

iii iv

B

ERp5 ADAM10

CTR CD30 i ii

iii iv

NKG2DL, ERp5 and ADAM10 expression in cHL ERp5 is expressed at the tumor site both by mesenchymal stromal cells and

Reed Sternberg cells

Mesenchymal stromal cells Reed Sternberg cells

100% MFI 147

100%

MFI 315

100%

MFI 116

100% MFI 151

87% MFI 65

90% MFI 62

90% MFI 47

100% MFI 315

100% MFI 116

97% MFI 136

Cell nu

mbe

r

Log red fluorescence intensity (a.u.)

HLA-‐I CD105 CD73 CD90

VIMENTIN COLLAGEN P4H TGII 0 20 40 60 80 100

MFI RAT

IO

B

0 5 10 15 20

MFI RAT

IO

39% MFI 6

R4

44% MFI 27

91% MFI 23

90% MFI 25

44% MFI 18 Ce

ll nu

mbe

r

2% 8% 42% MFI 20

13%


ULBP1 MIC-‐A ULBP2 ULBP3

CytoULBP1 CytoULBP2

39% MFI 9 R4

44% MFI 27

91% MFI 23

44% MFI 18

CytoULBP1 CytoULBP2

R4

44% MFI 27

91% MFI 23

44% MFI 18

CytoULBP1 CytoULBP2 CytoULBP4 CytoULBP3

ULBP4

1%

CytoMIC-‐A

i A

0 20 40 60 80

100

MFI RAT

IO ii

iii iv

i

iii

ii

0 5 10 15 20

MFI RAT

IO

iv

Phenotype and NKG2DL defective expression in cultured LNMSC from cHL.

NKG2DL, ERp5 and ADAM10 are expresssed in cultured LNMSC from cHL.

B

0 0.2 0.4 0.6 0.8 1 1.2

1.4 1.6 1.8 2 2.2

soluble molecule (ng/ml)


R4

33% MFI 10

93% MFI 32

1% 1%

R4

Cell nu

mbe

r

ERp5

CytoERp5 CytoADAM10

ADAM 10

A

0 2 4 6 8 10

ERp5 ADAM10

MFI RAT

IO 0 2 4 6 8 10

MFI RAT

IO

ERp5 ADAM10

iii

iv

iii

ERp5 and ADAM10 are expressed in the cytoplasm of cultured LNMSC from cHL, whereas NKG2DL are shed in the SN.

A

iv

ii i

iii

Log red fluorescence intensity (a.u.) Log far red

fluo

rescen

ce intensity

(a.u.)

NKG2DPE

CD8A

PC

Ex-‐vivo +IL15

+IL15+LNMSC +IL15+LNMSC+αTGFβ

B

LN + + + + IL15 -‐ + + -‐

LNMSC -‐ -‐ + -‐ an\-‐TGFβ -‐ -‐ -‐ +

0 25 50 75 100 125

CD8+NKG

2D (M

FI)

i

Vδ1+NKG

2D (M

FI)

0 25 50 75 100 125 ii

LN + + + + IL15 -‐ + + -‐

LNMSC -‐ -‐ + -‐ an\-‐TGFβ -‐ -‐ -‐ +

C

NKG

2D m

RNA (ΔC T) CD8αβ LN cells

Vδ1 LN cells

0 2 4 6 8 10

LN + + + + IL15 -‐ -‐ + +

LNMSC -‐ + -‐ +

Co-culture with LNMSC inhibits the upregulation of NKG2D induced by IL15 on γδ and αβ CD8 T cells

The inhibitory effect of LNMSC is reverted by anti-TGFβ mAbs LNMSC produce and release TGFβ

Autologous LNMSC + auto αβ or γδ T

D

0 100 200 300 400 500

TGFβ pg/ml ii

cytoTGF β

56%

MFI 18

LNMSC2

Cell nu

mbe

r i

A

medium +IL15

LNMSC+IL15 LNMSC

0

30

60 90

120 150

12h day

2 4 6 8

Vδ1 T cells

0 30 60 90

120 150 180

NKG

2D

expressio

n MFI

12h day

2 4 6 8

CD8 T cells i

ii

p<0.9770 C p<0.0002

p<0.0726

p<0.0001

0

50

100

150

10:1 20:1 40:1 80:1

p<0.9770 B p<0.0002

p<0.0726

p<0.0001

Vδ1 T cells : LNMSC ra2o

Vδ1 T cells only

basal 10:1 20:1 40:1 basal

0

50

100

150

CD8:LNMSC ra2o CD8 only

10:1 20:1 40:1 80:1

p<0.0001 p<0.0001

p<0.0167 p<0.637

NK

G2D

exp

ress

ion

MFI

0

50

100

150

CD8 only

10:1 20:1 40:1 80:1

p<0.0001 p<0.0001

p<0.0167 p<0.637

i ii

LNMSC mediated inhibition of NKG2D induced by IL15 on γδ and αβ CD8 T cells is ratio-dependent

The ratio between LNMSC and γδ or αβ CD8 T cells is important for the

inhibition of NKG2D expression

Inhibition is evident from day 4 of culture

RS773 6% MFI 24

NKG2D Fc Ce

ll nu

mbe

r

0 10 20 30

40 50

% CELL LYSIS

LNMSC αNKG2D

- -

+ -

+ +

- +

A

*

γδ T cells can kill RS773 lymphoma

cells expressing MIC-A

and ULBP3 via NKG2D

LNMSC inhibit the ability of γδ T cells to kill RS773 or C1R lymphoma cells

B RS773+VPA 20% MFI 22

NKG2D Fc

Cell nu

mbe

r

LNMSC αNKG2D

- -

+ -

+ +

- +

0

10 20

30 40

50

%CE

LL LYSIS

* ***

RS773

Surface ADAM10 ADAM10 shedding

ADAM10 shedding

Yellow square in B A B

Lymphoma cell

Treg cells

FDC mesenchymal stromal cell

l Th1 cell

NHL lymphoma microenvironment

Lymph node

Rela\ve mRN

A expressio

n

0

2

4

6

8

10

12

14

TGFbeta IL10 IFNgamma TNFalpha

Serie1

Serie2

Lymphomas non neoplas\c LN

IFNγ TNFα

IL10 TGFβ

Differentiation potential of LNMSC (alizarin: bone; oil red: adipocytes)

LN1 LN2 LN3 LN4 LN5 LN6 LN7 LNMSC expressed some characteristics of stem cells

Surface markers

NEG HLAI

ICAM1 CD146 CD90

CD73 CD105

PVR

Log Fluorescence intensity (a.u.)

Cell

num

ber

Cytoplasmic markers

NEG Collagen P4H

NEG ALP BSP Osteocalcin

Vimentin

Log Fluorescence intensity (a.u.)

Cell

num

ber

ACETYL-CoA CoA-SH

Acetoacetyl-CoA

Acetil-CoA

HYDROXYMETHYL GLUTARYL- COA

MEVALONATE 2NADPH+2H

2NADP+CoA-SH

5-Pyrophosphomevalonate

Dimethylallyl pyrophosphate

Geranyl pyrophosphate

Farnesyl pyrophosphate

Squalene

Squalene 2,3 epoxide Lanosterol CHOLESTEROL

Farnesyl pyrophosphate

+NADPH+H

2PPi+NADP

O2+NADPH+H

H2O+NADP

Isopentenyl pyrophosphate

PPi

Isopentenyl pyrophosphate

PPi

ATP ADP

ATP

ADP

ATP

ADP+Pi+CO2

Cholesterol synthesis fluvastatin

CoA-SH

geranylgeranyl pyrophosphate

Heme A, Dolichol,

Ubiquinon

Prenylated proteins (Rho,

Ras GTP-binding proteins

HMG-CoA reductase

Bisphosphonates

Zoledronic acid

19 reactions

Manumycin A, FTI and GTI inhibitors

ACAT-1,2 inhibitors (Avasimibe, K604,

Pyripyropene)

Acyl-CHOLESTEROL

TGFβ pg/ml/1

06 cellsl

i

0

50

100

150

200 C

ii

IL15 pg/ml/1

06 cells

0

40

60

80 100

20

* *

IL15

LNMSC

mRN

A (ΔC T)

0 2 4 6 8 10 12 *

B

-‐ -‐ -‐

-‐ -‐ +

+ + -‐

+ -‐ +

-‐ + -‐

Vδ2 Pam5 Zol1

+ -‐ -‐

TGFβ

-‐ -‐ -‐

-‐ -‐ +

+ + -‐

+ -‐ +

-‐ + -‐

Vδ2 Pam5 Zol1

+ -‐ -‐

** mRN

A (ΔC T) *

0 2 4 6 8 10 12 A LNMSC

*

IL10

* **

mRN

A (ΔC T)

0

2 4

6 8

10

12 D

-‐ -‐ -‐

-‐ -‐ +

+ + -‐

+ -‐ +

-‐ + -‐

LNMSC Pam5 Zol1

+ -‐ -‐

Vδ2 *

TNFα

mRN

A (ΔC T)

0

2 4

6 8

10

12 E Vδ2

-‐ -‐ -‐

-‐ -‐ +

+ + -‐

+ -‐ +

-‐ + -‐

LNMSC Pam5 Zol1

+ -‐ -‐

* IFNγ

mRN

A (ΔC T)

0

2 4

6 8

10

12 F

-‐ -‐ -‐

-‐ -‐ +

+ + -‐

+ -‐ +

-‐ + -‐

LNMSC Pam5 Zol1

+ -‐ -‐

Vδ2

LNMSC LNMSC

N-BPs prevent TGFβ and enhance IL15 production in LNMSC…

…reduce IL10 and stimulate TNFα or IFNγ in Vδ2 T lymphocytes

H

TNFα

pg/ml/1

06 cells **

Vδ2 I

IFNγ pg/m

l/106 cells

0

200

300

400

500

100

Vδ2

+LNMSC

*

+LNMSC

**

0

200

300

400

500

100

vs P815

**

% Cytotoxicity

*

0

10

20

30

40

50

60 *

Vδ2 E:T 10:1 Vδ2+LNMSC A

-‐ -‐ -‐ -‐

NKG2D CD16 Pam5 Zol1

+ -‐ -‐ -‐

+ -‐ -‐ -‐

+ -‐ -‐ +

-‐ + -‐ -‐

-‐ + -‐ -‐

-‐ -‐ -‐ -‐

+ -‐ + -‐

+ -‐ + -‐

+ -‐ -‐ +

***

B vs C1R

-‐ -‐ -‐

-‐ + -‐

% Cytotoxicity

0 10 20 30 40 50

60

*

*

Vδ2 E:T 10:1 Vδ2+LNMSC

RTX Pam5 Zol1

-‐ -‐ -‐

-‐ + -‐

+ -‐ -‐

+ -‐ -‐

-‐ -‐ +

-‐ -‐ +

i

vs C1R-‐MICA

% Cytotoxicity

0 10 20 30 40 50

60

**

Vδ2 E:T 10:1 Vδ2+LNMSC

*

*

RTX Pam5 Zol1

-‐ -‐ -‐

-‐ -‐ -‐

+ -‐ -‐

+ -‐ -‐

-‐ -‐ +

-‐ + -‐

-‐ + -‐

-‐ -‐ +

ii

LNMSC Pam5 Zol1

+ -‐ -‐

-‐ -‐ -‐

NKG

2D m

RNA (ΔC T)

0 2 4 6 8 10

+ + -‐

12

+ -‐ +

Vδ2

*

C **

D

NKG2D

Med

ium

+ Pam5

+ Zol1

NKG2D

CD16

NKG2D

Neg Ctr Vδ2 +LNMSC Vδ2

Med

ium

i

γδ T N

KG2D

+ (MFI)

0 25 50 75 100 125

LNMSC -‐ + + + +

an\-‐TGFβ -‐ -‐ -‐ -‐ +

*

**

Pam5 -‐ -‐ + -‐ -‐ Zol1 -‐ -‐ -‐ + -‐

ii Ce

ll nu

mbe

r

Log green fluo intensity (a.u.)

ADAM10 inhibitors

MSC B7-‐H1 PD-‐L1

X

NKG2DL

TGFβ, IDO and PGE2 synthesis inhibitors

Inac\va\on/elimina\on

of MSC by ADCC or CDC triggered with an\body specific

to lymphoma-‐associated MSC markers

An\body to B7-‐H1 (PD-‐L1) to avoid inhibi\on of T cells upon the

interac\on with the nega\ve regulator PD1

Inac2va2on of ADAM10 to reduce the release of NKG2DL

an\-‐ PD-‐L1 ab

TGFβ

IDO

PGE2

MSC

N-‐BPs zoledrobnate

TGFβ IDO

PGE2

IL15

TGFβ IDO

PGE2 IL15

Priming with N-‐BPs Iden2fica2on of other drugs to shic immunosuppression

to immune s2mula2on (induc2on of CD277 (BTN3A1) expression to trigger

Vδ2+ T cells)

A

B

ADAM10

CTL PD-‐1

FcγR+ effector cell

an\-‐MSC Ab

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LYMPHNODE&MESENCHYMAL&STROMAL&CELLS&(LNMSC)&& …