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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
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