Microenvironnement et Lymphome FolliculaireLymphome Folliculaire

Pr Karin TARTE, karin.tarte@univ-rennes1.frDES Hématologieh 2012

Unit U917MIcroenvironment and CAncer

MICAMICA

http://u917inserm.univ-rennes1.fr/

Normal B-cell differentiation

B-cell activation (STEP II)Ag-dependentRole of Th cells, myeloid cells

and stromal cells

BLOOD

SHM & CSRAID

B-cell lymphopoiesis (STEP I)Ag-independent (BCR dependent)Role of stromal cells

Humoral memory (STEP III)Long-lived plasma cellsMemory B cellsRole of stromal cells

BLOOD

VDJ recombinationRAG

FcRCR

MΦΦΦΦ/DC

ICAM-1

LFA-1

Step 1

CB DZ-FDCCXCR4

Dll1/Jg1

CD54/CD106

Step 3

Germinal Center

T-B boundary

CXCL12

BAFF, HGF, IL-15, IL-6

CXCL13

Dark zone

Naive B cell

HEV

FRC

CCL19CCL21

BCR

MRCSHM

CCR7hi

EBI2lo

CXCR5hi

CD40L CD40

TCRMHC II

CD4

Activated T cells

Activated B cells

CCR7Step 2 IL-2, IL-4

Dll1/Jg1

NotchNotch

CC

CXCR5

TFH

IL-21

CXCL13IL-4/IFN-γγγγ

CXCR5

CD40L

Step 4

CXCL13

Light zone

CSR

CCR7hi

BoneBone marrowmarrow

V(D)J rearrangement

t(14;18)

Naive

B cells

Germinal CenterGerminal Center

Somatic hypermutation

Pre-B

FL pathogenesis

Somatic hypermutation

Class switch recombination

FLLCFDCFDC

FRCFRC

MRC?MRC?

TAM

TFH

FL

Mourcin et al Front Immunol 2012;3:280Shaffer et al Annu Rev Immunol 2012; 30:565

LN or BM?CD20hiCD44loCD38pos

IgDnegCD138negCD34neg

FL LN

FL BM

� Moderated ttest P< 0.01; 696 PS� GSEA; MiSgdb signatures

16 ** *****

*

BRCA1

DNA repairCell cycle

TGF−−−−β β β β response

Nai

veB

Bgc

Mem

B

FL_LN

FL_BM

2

4

8

***

Nai

veB

Bgc

Mem

B

FL_LN

FL_BM

2

4

8

16** **

****

AID

LN AND BM

T4

FL microenvironment: immune cell

niches

T4

T4TCD8

TAM

T4

T4

T4

� Malignant B cells display some specificfeatures depending on their specificniches

� Intrinsic or due to differences betweenmicroenvironment composition?

FDCFDC

FRCFRC

MSCMSC

Stromal cellsImmune cells

T

MRCMRC

Microenvironment in FL

Tγδ

TCD8

NK

Treg 1 cancer1 cancer2 niches2 niches

Tumor nicheTumor niche

FL

Pre-FL

Normal B cell

TFH

TAM

How to study FL microenvironment?

LN, Tonsils

BMIHC/IF

FC

Whole tissue

TAMTFH

Functional analyses

Genomicapproaches

Dissociatedtissue

Purification of relevant

cell subsets

Lymphoma patientsHealthy donors

Classical T/NK compartments

Treg CCL22

TCD4

CD70

CD27

Th17NK

CD16

MICA/ULPB

CD137

Th1

TIM-3

Treg

TCD8

CCL22

Tγδ

NKG2DCXCR5

1. Malignant B-cells interfere with T-cellpolarization and activity2. B cells could be killed by activated γδT cellsand NK cells3. Prognostic markers?4. Therapeutic targets? (lenalidomide, antiCD137 agonist, γδ γδ γδ γδ T cell stimulation, ADCCCD16 dependent, anti-KIR, Treg inhibitors…)

Ramsay et al Blood 2009; 114:4713Yang et al Cancer Res 2006; 66:10145Yang et al Blood 2006; 107:3639Yang et al Blood 2007; 110:2537Yang et al Cancer Res 2009; 69:5522Weiyun et al Int J Cancer 2009; 124:239Braza et al J Immunol 2010; 184:134Catellani et al Blood 2008; 109:2078Kohrt, Houot et al Blood 2011; 117:2423Hilcheyi et al Blood 2011; 118:3591Yang et al JCI 2012;122:1271-82

CCR4

CD16TIM-3

Classical T/NK compartmentsCD8 number

Rab27AposCD3pos localization

T cells

GOOD

Alvaro et al. J Clin Oncol 2006; 24:5350 Ramsay et al. Blood 2009; 114:4713

Dave et al. N Engl J Med 2004; 351:2159

Macro

Granzyme B number

Laurent et al. Blood 2011; 118:5371

GOOD

BAD

What about TFH

FL_ENV RLN_ENV

0

20

40

60

% CXCR5hiICOShiamong

CD4posT cells

RLN TONS FL DLBCL

****** ***

P<0.01

TFH signature

FL_ENV signature FL_Env RLN_EnvSNR

Strong TFH infiltration in FLFL-TFH display a specific GEP

FL

TF

HF

L T

FH

FL

TF

HF

L T

FH

To

ns

TF

HT

on

s T

FH

To

ns

TF

H

To

ns

TF

H

FL TFH: more than TFH

30.6±±±±5% 14.4±±±±4%

IL-4

PD-1

FL TONSIL4

mR

NA

Fo

ld

Ch

an

ge

**

0

200

400

FL TFH overexpress functional IL-4

IL-4

PP--STAT6STAT6//CD5CD5

PP--STAT6STAT6//PDPD--11

Tonsil/RLNPSTAT6PSTAT6

FLPSTAT6PSTAT6

FL

FL TFH: more than TFH

*

CD40LG

mR

NA

Fo

ld

Ch

an

ge

0

5

10

+ TnonFH + TFH

40% 15%45%

B cells

Spontaneous apoptosisDrug-induced apoptosis

1p36.32

Active Caspase 3

TFH

IL-4

CD40L

P-STAT6

TNFLT

IFNγγγγ

FDCFDC

FRCFRC

MRCMRC

TAMPD-1

1p36.32

BTLA

HVEM

Calvo et al Blood 2008; 112:3818Xerri et al Human Pathol 2008; 39:1050Pangault Leukemia 2010 ; 24:2080Launay Leukemia 2011, 26:559Amé-Thomas Leukemia 2012, 26:1053

PD-L1?

1. FL TFH display some specific features2. Direct B-cell supportive activity3. Indirect protumoral activity4. Link with genetic alterations?5. Prognostic marker?6. Therapeutic target (anti-PD1)?

FL TFH: more than TFH

CXCR5CD25

Foxp3

14%

15

20

Foxp3

+in

ICOShi

***

Specific infiltration by TFH/Treg cellsOrigin?

ICOS

0

5

10

15

% CD25+Foxp3

CD4+CXCR5hi ICOS

Foxp3Foxp3//ICOSICOS

FL TONS

TONS FL

TFR

0 5 100

20

40

60

TF

H

R=0.78

Human TFR: a new FL-specific cell subset

Teff

mR

NA

Fo

ld

Ch

an

ge

FOXP3500

250

10

0

FL

TF

RF

L T

FR

FL

TF

RT

reg

Tre

gT

reg

FL

TF

HF

L T

FH

FL

TF

HF

L T

FH

To

ns

TF

HT

on

s T

FH

To

ns

TF

H

To

ns

TF

H

T-cell inhibition

Foxp3 number

CFSE

Teff

Teff + TFH Teff + TFR

Treg

Identification of human TFRRole in B-cell lymphomas?

inhibitionCarreras et al. Blood 2006; 108:2957

Foxp3 localization

Farinha et al. Blood 2010; 115: 289

Myeloid compartment

T cells

LN CD68 numberCHOP R-CHOP

GOOD

Dave et al. N Engl J Med 2004; 351:2159

Macro

Farinha et al. Blood 2005; 106:2169 Taskinen et al. Clin Cancer Res 2007;13:5784

PB monocyte count

Wilcox et al. Leuk Lymphoma 2012; online

GOOD

BAD

BCR-dependent Ag-independent signaling

MΦC-type lectin

Follicular lymphomas: importance of BCR signaling

VVVVHHHH VVVVLLLL

oligomannose

Somatic hypermutations

New sites ofN-glycosylation

VVVVHHHH

VVVVLLLLOligomannose

FL

VVVVLLLL

CCCCHHHH

CCCCLLLL

DC

Stromal cells

Myeloid compartmentC-type lectin

Oligomannose

CD32CD64

Coelho et al PNAS 2010; 107:18587Epron et al Leukemia 2012; 26:139Clear et al Blood 2010; 115:5053Schwaller et al Blood 2007; 109:331Novak et al Cancer Res 2009; 69:4217Hildebrand et al J Exp Med 2010; 207:2569Lin et al Blood 2011; 117:872Chao et al Cell 2010; 142:699

TNFSF13B

polymorphism

TFHCD40L

CD40

IL-15/

IL-15Rββββ/

γγγγc

P-

STAT5

BAFF

MO

CEC

MDSC

Angiogenicfactors

CD64

SIRPαααα

CD47

TAM

1. Myeloid cell phenotypic and functionalheterogeneity (supportive & inhibitoryactivities)2. Therapeutic targets (inhibitors of BCRsignaling, inhibitors of IDO/arginase, antiCD47, antibodies with increased affinity forCD32, inhibitors of angiogenesis…)?

SR

IL-15/

IL-15Rαααα

TAM

γγγγc

CD163

TNFRSF13C

mutation

Functional heterogeneity of stromal microenvironment

Survival

FL B cell

MSC

FRC-like

Proliferation

T cell

TNF + LT

TNF + LT+ IFN-γγγγ

IDO

CD40L

IL-2IL-4

IFN-γγγγ

FRC-like

Ame-Thomas Blood 2007; 109:693El Hajjami Cancer Res 2009; 69:3228

Direct supportive activity of FL stromal cells

30

***

** **

cp

m)

P1 MSC

Guilloton Blood 2012 ; 119:2556

B-cell line

0

10

20

30

CTRL +TNF/LT CTRL +TNF/LT

HD-MSC FL-MSC

B cellsalone

3H

-Td

R in

co

rpo

rati

on

(x10

3cp

m)

FL patientsNormal donors

(matched)

FL-MSC exhibited a significantly better B-cell supportive activity than HD-MSC

Stromal cells as organizers of FL cell niche

FL patients

No t(14;18)No karyotypic abnormality

P1 MSC

FL-MSC signature320 upregulated genes60 downregulated genes

95 PS 408 PS 513 PS

Mann-WhitneyP < .05

SAMFDR < 5%

0

1

2

3

4

5

6***

CCL2

FL patientsNormal donors

(matched)

Affymetrix U133 2.0

abnormality 60 downregulated genes

Quantification in BM plasma

HD FL

CCL2 (pg/ml)

HD

-MSC

FL-MSC

Specific FL-MSC signatureOverexpression of CCL2 by FL-MSCCCL2 is present within FL cell niche

HD FL1

10

100

1000

10000 ***

0

5

10

15

20

25 *

supernatant

CCL2 depleted supernatant

CCL2 depleted supernatant+rhCCL2

*

Monocyte

specific migration (%)

HD-MSC FL-MSC

w/o MSCwith MSC

1

10

100 *****

ND

TNF IL-101

10

100

*

*

*

Fo

ld c

han

ge u

po

nL

PS

sti

mu

lati

on

TNF IL10 IL12A

Co

ncen

trati

on

(p

g/m

L)

TAM-like phenotype

Monocyte recruitment

IL-15/

IL-15RααααTAM

0 50000 100000 150000 200000

Alone

+Macrophages

+MSC

+MSC+Macrophage

**

***

Thymidine incorporation (cpm)

B-cell growth

Fold change of CCL2 production/untreated MSC

0.1 1 10 100 1000

+FL B cells

+BL2

+VAL

+RL

+TNF/LT

ns

*

*

*

***

Stroma modification byFL B cells P-

STAT5

IL-15RααααTAM

IL-15Rββββ/

γγγγc

Stromal compartmentAme-Thomas et al Blood 2007; 109:693Guilloton et al Blood 2012; onlineLwin et al Leukemia 2009; 23:170Lwin et al Blood 2010; 116:5228Singh et al. Oncogene 2011; 30:4874

VCAM-1

CXCL12/CXCL13

BAFF, Hh

HGF, IL-15

IL-4

TFH

P-

STAT6

TNF, LT

IFNγγγγ

CCL2

TNF/LT

CXCR4

CXCR5

P<.01

FRC signature

Migration Index

w/o AMD3100

with AMD3100

1

10

100

**

**

CXCL12 MSC

1. Stromal cells recruit and support directlymalignant B cells2. Malignant B cells confer a supportive phenotypeto stromal cells3. Stromal cells organize malignant B-cellniche (interactions with TAM, T cells, neutrophils…)4. Specific therapeutic targets ?

FL B cell

STAT6

TFH

CD40L

CD40

P-

STAT5

IL-15/

IL-15Rαααα

TAM

IL-15Rββββ/

γγγγc

TNF, IL4

IFNγγγγ

FL-MSC HD-MSC

Cibler le microenvironnement?

� Ciblage TFH (PD-1/PD-L1 ?)

� Ciblage Treg (CCR4 inhibiteurs?)

� Ciblage cellules cytotoxiques (anti-CD137, anti-KIR, Ac optimisés pour ADCC, agonistes Tγδ?)

� Ciblage macrophages (anti-CD47, inh CSF1-R ?)� Ciblage macrophages (anti-CD47, inh CSF1-R ?)

� Inhibition de l’interaction avec cell. stromales� anti-VLA-4 Natalizumab

� Btk inhibitor PCI-32765

� CXCR4 inhibitors� AMD3100 (perixlafor)

� Pepducins (small inhibitory lipopeptides)

� Lenalidomide (decrease of CXCL12 synthesis)

� PCI-32765 (inhibition of chemokine-induced signalling) 24

Conclusions/Perspectives� Le FL = une histoire de niche

� Nécessité d’études biologiques fonctionnelles poussées adossées aux essais thérapeutiquespoussées adossées aux essais thérapeutiques

� Intégrer les nouvelles données de la physiopathologie dans la démarche thérapeutique

� Ne pas considérer que la génétique, ne pas opposer génétique et immunologie