Biomarqueurs et rejet chronique pulmonaire: Résultats de l’Initiative de la Cohorte

de Greffe Pulmonaire (COLT)

Antoine Magnan, Adrien Tissot, Aurore Foureau, Maxim Durand, Carole Brosseau, Pierre-Joseph Royer,

Eugénie Durand, Richard Danger, Sophie Brouard

Université de Nantes, CHU de Nantes, l’institut du thorax, Service de pneumologie,

INSERM 1087 / CNRS 6291, INSERM UMR 1064

CLAD : mécanismes

Royer PJ et al., transplantation 2016

COLT

Inclusion Transplantation

V0 V1 V2 5 years

Chronic rejection

V3

samples

Research of early predictive factors of CLAD Towards 4P medicine ?

A Magnan A Tissot

M Stern

O Brugière (Bichat), V Boussaud (HEGP)

S Mussot

JF Mornex

C Dromer

C Pison

R Kessler

M Dahan

C Knoop

C Benden

L Nicod JD Aubert

M Reynaud-Gaubert

COLT

COLT / SysCLAD Story

Recherche Biomédicale Recherche Non Interventionnelle

500°

1200°

07 2017: Inclusions: 1804

Transplanted: 1451

2008: LT program VLM / AGL

2009: PHRC National

INSERM R Pays de Loire

ABM Novartis, Sanofi,

Astellas

Dysfonction Chronique du Greffon:

Classification of adjudicated patients after 3 years post transplant

• Nov 2017: 1st adjudications at 5 years: 250 patients « stable » at 3 years

Do

no

r’s

Bro

nc

hia

l

ep

ith

elial

cells (

BE

C)

Recip

ien

t’s

Imm

un

e c

ells

Modification of BEC profile

Airway epithelial cells exposed to allogeneic T cells produce MMP9 through a CCL2/CCR2 pathway: implications for chronic lung allograft dysfunction.

Pain M, Royer PJ, AJT 2016

MMP9 Story

0.0

0.3

0.6

0.9

1.2

TL

Monocytes

-

+

+ +

--

Rela

tive e

xpre

ssio

n (

2-

CT

)

0

20

40

60

80 ***

*

TL

Monocytes

-

+

+ +

--

Rela

tive e

xpre

ssio

n (

2-

CT

)

0

20

40

60

***

***

TL

Monocytes

-

+

+ +

--

Rela

tive

exp

ress

ion (

2-

CT

)

0.0

0.3

0.6

0.9

1.2

Without TGF-1

With TGF-

TL

Monocytes

-

+

+ +

--

Rela

tive e

xpre

ssio

n (

2-

CT

)

A B

+ TGF-1

0

100

200

300

400

500

Without TGF-

With TGF-

*

*

TL

Monocytes

-

+

+ +

--

MM

P-9

(ng/m

L)

C

ZO-1 E-Cadherin

Fibronectin MMP-9 MMP-9

ZO-1

E-Cadherin

Fibronectin

-Actin

TL

Monocytes - - +

- + +

- - +

- + +

Airway epithelial cells exposed to allogeneic T cells produce MMP9 through a CCL2/CCR2 pathway: implications for chronic lung allograft dysfunction.

Pain M, Royer PJ, AJT 2016

-18 -12 -6 VCLAD +6 +12 + 180

300

600

900

1200

BOS

Stable

*

RAS

MM

P9 (

pg/m

L)

0 20 40 60 80 100

0

20

40

60

80

100

Area:0.8561

p<0.0001

100%-specificity

Sensi

tivi

ty %

A B

Stable BOS

10

100

1000

10000

ng/m

l

C

A cut-off value of 139 ng/ml of MMP-9 allowed the prediction of BOS at 6 months with 94% sensitivity and 73% specificity

Pain M, Royer PJ, AJT

Airway epithelial cells exposed to allogeneic T cells produce MMP9 through a CCL2/CCR2 pathway: implications for chronic lung allograft

dysfunction.

Regulatory T cells in transplantation

• Regulation of inflammation : asthma, autoimmune diseases, …

• Growing interest in studying T regs in organ transplantation : operational tolerance, acute

rejection, clinical trials.

• No consensus about the link between Tregs and BOS occurrence.

Befor

e Tp

1 to 6

mth

after

Tp

6 mth

befor

e CLA

D CLAD

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

% T

regs

among C

D4

T C

ells

**

Regulatory T cells profile

STA BOS

4 Regulatory CD4 T Cells

STABO

S0

1

2

3

4

5

6

7

8

% T

regs

among C

D4

T C

ells

1 to 6

month

s af

ter

TP

***

CD4

Co

un

t

FoxP3

CD

25

1

3

2

4

STABO

S0.00

0.02

0.04

0.06

0.08

0.10

% T

regs/

CD

4 C

D25-

among T

Cel

ls

1 to 6

month

s af

ter

TP

***

STABO

S0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.20

% T

regs/

CD

8+

am

ong T

Cel

ls

1 to 6

month

s af

ter

TP

*

Regulatory T cells profile

STABOS

0

200

400

600

800

1000

1200

1400

MF

I C

D39 o

f T

regs

1 to 6

month

s af

ter

TP

CD39

STABO

S0

40

80

120

160

200

240

280

320

360

400

MF

I C

D73 o

f T

regs

1 to 6

month

s af

ter

TP

STABO

S0

500

1000

1500

2000

2500

3000

MF

I C

D15s

of

Tre

gs

1 to 6

month

s af

ter

TP

CD73 CD15s

BOS

STA

0

2000

4000

6000

8000

MF

I C

D25 o

f T

regs

1 to 6

month

s af

ter

TP

BOS

STA

0

1000

2000

3000

4000

MF

I C

D39 o

f T

regs

1 to 6

month

s af

ter

TP

BOS

STA

0

5000

10000

15000

20000

25000

MF

I P

D1 o

f T

regs

1 to 6

month

s af

ter

TP

BOS

STA

0

2000

4000

6000

8000

MF

I C

D15s

of

Tre

gs

1 to 6

month

s af

ter

TP

CD25 CD39 PD1 CD15s

FoxP3

CD

45

RA

1. FoxP3 CD4+ Tregs

1.1 nTregs FoxP3low CD45RA+

1.2 nonTregs FoxP3low CD45RA-

1.3 mTregs FoxP3hi CD45RA-

1.1

1.2

1.3

STA

BOS

0.0

0.5

1.0

1.5

2.0

2.5

% m

Tre

gs

among C

D4

T C

ells

1 to 6

month

s af

ter

TP

*1.3

Conclusion

• Increase of Tregs proportion with a memory phenotype early after TP for patients who

will declare a BOS in the five years.

0 10 20 30 40 50 600

20

40

60

80

100

Monts post TP

BO

S f

ree

surv

ival

(%

)

Log rank p = 0,0035

**

Tregs among CD4 < 2,08 %

Tregs among CD4 > 2,08 %

• New potential biomarker of the BOS occurrence, which could help to manage CLAD

after lung transplantation in the next decades.

0 10 20 30 40 50 60 70 80 90 1000

10

20

30

40

50

60

70

80

90

100

100% - Specificity%

Sen

sitivity%

Circulating CD9+ B cells: biomarker of long-term BOS-free survival after Lung Transplantation (94/100) Carole Brosseau 1,2,5,6 †, Maxim Durand 1,2,3†, Eugénie Durand 1,2 , Richard Danger 1,2, Jennifer Loy 5,6, Aurore Foureau 5,6,, Mélanie Chesneau 1,2, Philippe Lacoste 5,6, Pierre-Joseph Royer 5,6, Adrien Tissot1,2,3,5,6, Antoine Roux5, Martine Reynaud-Gaubert6, Romain Kessler7, Sacha Mussot8, Claire Dromer9, Olivier Brugière10, Jean François Mornex11, Romain Guillemain12, Marcel Dahan13, Christiane Knoop14, Christophe Pison15, Laurent Nicod16, Antoine Magnan 5,6$, Sophie Brouard 1,2,4$ & COLT and SysCLAD consortia*.

CD

24

CD38

Doublet &

dead cells

exclusion

SS

C-A

SS

C-A

FSC-A CD19

CD

27

CD22

A

B B Lymphocytes Memory cells Naive cells

Plasma cells Transitional cells

% o

f C

D19+

cells

% o

f m

em

ory

cells

% o

f naiv

e c

ells

% o

f pla

msa

cells

% o

f tr

ansitio

nal cells

Months Months Months

Months Months

CD19

0 1 6 12 18 24 300

5

10

15

20

STA

BOS - rejet

BOS + rejet

HV

Months

% o

f C

D19+

B c

ell

s

mémoires

0 1 6 12 18 24 300

10

20

30

40STA

BOS - rejet

BOS + rejet

Legend

naives

0 1 6 12 18 24 30

40

60

80

100

STA

BOS - rejet

BOS + rejet

Legend

plasmo

0 1 6 12 18 24 300.0

0.5

1.0

1.5

2.0

2.5STA

BOS - rejet

BOS + rejet

Legend

transi

0 1 6 12 18 24 300

2

4

6

8STA

BOS - rejet

BOS + rejet

Legend

CD19

0 1 6 12 18 24 300

5

10

15

20

STA

BOS - rejet

BOS + rejet

HV

Months

% o

f C

D19+

B c

ell

s

CD19

0 1 6 12 18 24 300

5

10

15

20

STA

BOS - rejet

BOS + rejet

HV

Months

% o

f C

D19+

B c

ell

s

***

Lymphocyte CD19+

Transitional

Plas

ma

cells

Memory

Naive

T3 dans CD19

0 1 6 12 18 24 300.0

0.2

0.4

0.6

0.8

STA

BOS - rejet

BOS + rejet

HV

T2 dans CD19

0 1 6 12 18 24 300

1

2

3

4STA

BOS - rejet

BOS + rejet

HV

T1 dans CD19

0 1 6 12 18 24 300

1

2

3

4

STA

BOS - rejet

BOS + rejet

HV

CD

27

CD22

CD

24

CD38

IgD

IgM

Transitional

T1/T2 T3

Months

% o

f T

3

am

ong t

ransitio

nal cells

Months

% o

f T

1/T

2

am

ong t

ransitio

nal cells

Months %

of

CD

27+

non

-conventio

nal

am

ong t

ransitio

nal cells

% o

f T

3

am

ong

CD

19+

cells

% o

f T

1/T

2

am

ong

CD

19+

cells

Months Months Months

% o

f C

D27+

non

-conventio

nal

am

ong C

D19+

cells

B

** *** *** *

CD27+

non-

convention

al

T1 dans transi

0 1 6 12 18 24 300

20

40

60

80STA

BOS - rejet

BOS + rejet

HV

T2 dans transi

0 1 6 12 18 24 300

20

40

60

80

STA

BOS - rejet

BOS + rejet

HV

T3 dans transi

0 1 6 12 18 24 300

20

40

60

80

STA

BOS - rejet

BOS + rejet

HV

CD19

0 1 6 12 18 24 300

5

10

15

20

STA

BOS - rejet

BOS + rejet

HV

Months

% o

f C

D19+

B c

ell

s

CD19

0 1 6 12 18 24 300

5

10

15

20

STA

BOS - rejet

BOS + rejet

HV

Months

% o

f C

D19+

B c

ell

s

% o

f C

D9+

cells

Months

**

CD9

0 1 6 12 18 24 300

5

10

15

STA

BOS - rejet

BOS + rejet

Legend

CD9 dans transi

0 1 6 12 18 24 300

20

40

60

80

100

STA

BOS - rejet

BOS + rejet

Legend

transi CD9 dans 19

0 1 6 12 18 24 300

1

2

3

4

STA

BOS - rejet

BOS + rejet

Legend***

% o

f C

D9+

am

ong

tra

nsitio

nal cells

% o

f tr

ansitio

nal C

D9+

am

ong

CD

19+

cells

CD19

0 1 6 12 18 24 300

5

10

15

20

STA

BOS - rejet

BOS + rejet

HV

Months

% o

f C

D19+

B c

ell

s

CD19

0 1 6 12 18 24 300

5

10

15

20

STA

BOS - rejet

BOS + rejet

HV

Months

% o

f C

D19+

B c

ell

s

T1/T2 CD27+ non-conventional T3 CD9+

Blood Gene Expression Predicts

Bronchiolitis Obliterans Syndrome Appearance

After Lung Transplantation

Richard Danger*, Pierre-Joseph Royer*, Damien Reboulleau, Eugénie Durand, Jennifer Loy, Adrien Tissot, Philippe Lacoste, Antoine Roux,

Martine Reynaud-Gaubert, Carine Gomez, Romain Kessler, Sacha Mussot, Claire Dromer, Olivier

Brugière, Jean-François Mornex, Romain Guillemain, Marcel Dahan, Christiane Knoop, Karine

Botturi, Christophe Pison, Angela Koutsokera, Laurent P. Nicod,

Sophie Brouard*, Antoine Magnan* and the COLT and SysCLAD Consortia

• Enrichment of down-regulated and immune-related genes in PRED versus STA comparison

number of genes within the enriched ontology

Gene ontology analysis:

Blood gene expression associated with BOS

• Identification of differential genes (t-statistic from R limma package; p.value <5% and fold change >1.5)

Prediction of BOS: independent validation

• Validation of genes associated with BOS appearance

-individual qPCR -samples from new patients: n= 13 STA & 11 PRED independent validation

POU2AF1: POU class 2 associating factor 1

BLK: B lymphoid tyrosine kinase

TCL1A: T-cell leukaemia/lymphoma 1A

Stable during time

Prediction of BOS: independent validation

• Validation of genes associated with BOS appearance

• Prediction of BOS appearance

-individual qPCR -samples from new patients: n= 13 STA & 11 PRED independent validation

3 genes to predict BOS

AUC= 0.83 AUC= 0.77 AUC= 0.78 ROC curves: Excellent discriminative ability

POU2AF1: POU class 2 associating factor 1

BLK: B lymphoid tyrosine kinase

TCL1A: T-cell leukaemia/lymphoma 1A

Blood Gene Expression Predicts Bronchiolitis Obliterans Syndrome Appearance

After Lung Transplantation

Validation of these 3 genes in a large prospective cohort

• Identification of 3 genes as predictive biomarkers of BOS • Whole blood and qPCR: non-invasive and compatible with clinical settings • Suggests a role of B cells in BOS mechanisms

Conclusion

patent: EP16306125.2 work submitted

Conclusions: Biomarqueurs issus de COLT et SysCLAD

• MMP9

• Treg

• B transitionnels CD9 +

• Pou2AF1, BLK, TCL1A

A valider en pratique clinique Score composite à construire

Sophie BROUARD Carole BROSSEAU Maxime DURAND Richard DANGER

CRTI – INSERM UMR 1064

Institut du thorax – INSERM UMR 1087

Antoine MAGNAN Philippe LACOSTE Adrien TISSOT Carole BROSSEAU Eugénie DURAND Jennifer LOY Pierre-Joseph ROYER Aurore FOUREAU

Thank you for your attention

COLT Consortium

Flow Cytometry Platform

Remerciements

PHRC 2009