Non-HLA anti-endothelial cell antibodies: an old problem ... · Non-HLA anti-endothelial cell...
Transcript of Non-HLA anti-endothelial cell antibodies: an old problem ... · Non-HLA anti-endothelial cell...
Non-HLA anti-endothelial cell antibodies:
an old problem with new insights
Necker - Enfants Malades
Prof Dany Anglicheau, MD PhD
Department of Nephrology and Renal Transplantation
Necker Hospital, Paris
France
The acute rejection phenotypes
Two prototypic types of acute rejection
The acute rejection phenotypes: the conventional view
T cell
NK cell
Plasma cell
Monocyte
Interstitial inflammation Tubulitis Glomerulitis Peritubular capillaritis
Transplant glomerulopathy
C4d staining
B cell
T cell mediated
Antibody-mediated
Anti-HLA donor specific antibodies
Day-3 Month-2 Month-12
32 year old manSecond graft for IgANLiving donor (HLA-identical brother)
0 30 60 90 120 150 180 210 240 270 300 330 360 390 420 450 480 510 540 5700
200
400
600
800
Biopsie
Ñ
Ñ Solumedrol
ÑÑ
Ä
Ä Rituximab
Ä
IVIG 2 g/kgEchanges plasmatiques
ÑÑÑ ÑÑ
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Délai post-transplantation ( jours)
Cré
atin
iné
mie
(m
mo
l/l)
Seru
mC
reat
inin
e(m
mo
l/L)
Time post-transplantation (days)
Biopsy
0 30 60 90 120 150 180 210 240 270 300 330 360 390 420 450 480 510 540 5700
200
400
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Biopsie
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Ñ Solumedrol
ÑÑ
Ä
Ä Rituximab
Ä
IVIG 2 g/kgEchanges plasmatiques
ÑÑÑ ÑÑ
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Délai post-transplantation ( jours)
Cré
atin
iné
mie
(m
mo
l/l)
Steroid pulses
0 30 60 90 120 150 180 210 240 270 300 330 360 390 420 450 480 510 540 5700
200
400
600
800
Biopsie
Ñ
Ñ Solumedrol
ÑÑ
Ä
Ä Rituximab
Ä
IVIG 2 g/kgEchanges plasmatiques
ÑÑÑ ÑÑ
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Délai post-transplantation ( jours)
Cré
atin
iné
mie
(m
mo
l/l)
2 g/kg IVIG
0 30 60 90 120 150 180 210 240 270 300 330 360 390 420 450 480 510 540 5700
200
400
600
800
Biopsie
Ñ
Ñ Solumedrol
ÑÑ
Ä
Ä Rituximab
Ä
IVIG 2 g/kgEchanges plasmatiques
ÑÑÑ ÑÑ
............................ ........................................................
Délai post-transplantation ( jours)
Cré
atin
iné
mie
(m
mo
l/l)
Plasmapheresis
0 30 60 90 120 150 180 210 240 270 300 330 360 390 420 450 480 510 540 5700
200
400
600
800
Biopsie
Ñ
Ñ Solumedrol
ÑÑ
Ä
Ä Rituximab
Ä
IVIG 2 g/kgEchanges plasmatiques
ÑÑÑ ÑÑ
............................ ........................................................
Délai post-transplantation ( jours)
Cré
atin
iné
mie
(m
mo
l/l)
Rituxan
0 30 60 90 120 150 180 210 240 270 300 330 360 390 420 450 480 510 540 5700
200
400
600
800
Biopsie
Ñ
Ñ Solumedrol
ÑÑ
Ä
Ä Rituximab
Ä
IVIG 2 g/kgEchanges plasmatiques
ÑÑÑ ÑÑ
............................ ........................................................
Délai post-transplantation ( jours)
Cré
atin
iné
mie
(m
mo
l/l)
Month-20
Natural history of ABMR recapitulated in the
absence of anti-HLA DSA!
Delville M, Anglicheau D. Hum Immunol. 2016 Nov;77(11):1055-1062.
A single case
Non-HLA Donor/Recipient mismatches: emerging data
Reindl-Schwaighofer R et al. Lancet. 2019 Mar 2;393(10174):910-917
Mesnard L et al. PLoS Comput Biol. 2016 Sep 29;12(9):e1005088.
Stapleton CP et al.Am J Transplant. 2019 Feb 27. doi: 10.1111/ajt.15326.
Steers NJ et al.New Engl J Med 2019;380:1918-28
Jackson AM, …Anglicheau D. Human Immunol 2019
Development of non-HLA antibodies
Vascular injury can initiate AECA development or, alternatively, AECAs may elicit new or contribute to ongoing vascular injury or dysfunction potentiating the release of pro-inflammatory exosomes leading to
broader immune activation
Identification of agonistic IgG against angiotensin II type 1 receptor (AT1R) in HLA-matched patients with vascular rejection
Patient’s anti-AT1R Abs
Dragun D et al. NEJM 2005
Functional AT1R Abs act as an allosteric receptor agonist by binding to the second extracellular loop of AT1R and initiating biological processes leading to graft injury
33 KTx Pts with refractory AMR
13 (39%) pts had HLA DSA+
16 (48%) pts had AT1R+ without HLA Abs
AT1R Abs as a major culprit
940 patients between 2008 and 2012 (Nantes, Lyon, Necker University
Hospitals)
Adults recipients
kidney or a combined kidney and pancreas transplantation
Heart beating deceased donor
All patients were under CNI and MMF with an induction therapy
HLA sensitization assessed by Luminex
Surveillance biopsies within the first year of follow-up to identify subclinical ARE
Deltombe C et al. Transpl Int. 2017
Pre-Transplant AT1R Abs
…a multicenter study
Deltombe C et al. Transpl Int. 2017
Clinically relevant TCMR Clinically relevant ABMR Death censored Graft
survival
Pre-transplant AT1R Abs: a multicenter study
Post-transplant AT1R Abs
Kidney Int. 2019 Mar 15. pii: S0085-2538(19)30173-5.
1845 kidney transplant recipients assessed simultaneously for the presence of circulating anti-AT1R antibodies and kidney allograft histology within the first year after transplantation
in whom 299 (16.2%) showed histologic features of active antibody-mediated rejection according to the Banff classification (g+ptc > 1).
Among patients with histologic features of active antibody-mediated rejection: 51 (17.0%) had anti-AT1R antibodies, 147 (49.2%) had donor-specific anti-HLA antibodies (DSAs), 75 (25.1%) had both antibodies, 26 (8.7%) had no antibody.
Post-transplant AT1R Abs
Kidney Int. 2019 Mar 15. pii: S0085-2538(19)30173-5.
Post-transplant AT1R Abs
Kidney Int. 2019 Mar 15. pii: S0085-2538(19)30173-5.
Expression levels of endothelial-associatedtranscripts (ENDATs) in patients withfeatures of active antibody-mediatedrejection according to anti-AT1R antibodyand DSA status.
See S et al. J Am Soc Nephrol 29: 1761–1770, 2018.
Day 0 12 months
Tx
Serum
Acute rejection
We assessed the generation of IgG Nabs reactive to malondialdehyde (MDA) during the first year following transplantation
in 635 patients transplanted at Necker Hospital, Paris, France.
Polyreactive autoantibodies: The Necker cohort
Natural antibodies often display a polyreactive profile in that they react to multiple, distinct antigenic structures as well as apoptotic cells.
Polyreactive autoantibodies produced by B-cell clones can bind apoptotic cells and activate complement.
Such autoantibodies have the potential to amplify microcirculation injury caused by alloantibody in antibody-mediated transplant rejection.
Polyreactive autoantibodies: The Necker cohort
See S et al. J Am Soc Nephrol 29: 1761–1770, 2018.
Biopsies at 1-year post-transplant or at time of rejection within the first year
Nabs+: development of post-transplant Nabs (definedas 50% increase in reactivity to malondialdehyde)
Polyreactive autoantibodies: The Necker cohort
See S et al. J Am Soc Nephrol 29: 1761–1770, 2018.
Biopsies at 1-year post-transplant or at time of rejection within the first year
Risk factors associated with
graft loss in univariate and
multivariable analyses.
Determinants of graft loss: univariate and multivariate analysis
See S et al. J Am Soc Nephrol 29: 1761–1770, 2018.
Polyreactive Abs
increase is an
independent risk
factor of graft loss.
Inclusion criteria: 1st or retransplantation
Deceased or living donor
Acute dysfunction or delayed graft function
Biopsy proven acute rejection with significant microcirculation inflammation (i.e.g+ptc ≥3)
During the first 3 months post transplantation
No HLA DSA by luminex SA assay (A/B/Cw/DR/DQ/DP)
Identification of early acute rejections with features of ABMR in the absence of HLA DSA assessed using Luminex® SAB.
French (and Belgian) nation-wide study: 22 centers
Day 0 3 months
Tx
Serum AMVR (Acute MicroVascular Rejections)
Acute rejection
Looking for new targets: a French nation-wide study
Delville M, Lamarthee B, …, Anglicheau D. J Am Soc Nephrol. 2019 Apr;30(4):692-709)
gptc
vC4
di t
cg ci ct cv ah0
1
2
3
Elementary Lesions
Banff S
core
(mean
±S
EM
)
0
1
2
3
4
5
6
Individual Cases
g+
ptc
score
gptc
A.# B.#
E.#
G.# H.#
F.#
D.#C.#
gptc
vC4
di t
cg ci ct cv ah0
1
2
3
Elementary Lesions
Banff S
core
(mean
±S
EM
)
0
1
2
3
4
5
6
Individual Casesg+
ptc
score
gptc
A.# B.#
E.#
G.# H.#
F.#
D.#C.#
gptc
vC4
di t
cg ci ct cv ah0
1
2
3
Elementary Lesions
Banff S
core
(mean
±S
EM
)
0
1
2
3
4
5
6
Individual Cases
g+
ptc
score
gptc
A.# B.#
E.#
G.# H.#
F.#
D.#C.#
Pathological characteristics of the early acute microvascular rejections
Mean g+ptc score: 3.9±0.25
Delville M, Lamarthee B, …, Anglicheau D. J Am Soc Nephrol. 2019 Apr;30(4):692-709)
Assessment of known AECAs
Assessment of of anti-angiotensin type 1 receptor (anti-AT-1R), anti-endothelin-1 type A (anti-ETAR) and natural polyreactive antibodies
No clear increase of known AECAs in AMVR serum (anti-MICA detected in 2/20 AMVR sera)
0 5 10 15 200
750
1500
2250
3000
Anti-ETAR Abs (UI/mL)
Re
ativity t
o M
DA
by D
EL
FIA
(U
I/m
L)
r2=0.26
P=0.0065
A.# B.#
Anti-ETAR Abs Anti-AT1R Abs
0
5
10
15
20
U/m
L
AMVR
StableP=0.39 P=0.14
C.# D.#
Reactivity to apoptotic cells
by FACS
Reactivity to MDA by
DELFIA
AMVR
StableAMVR
Stable
0 5 10 15 200
5
10
15
20
Anti-ETAR Abs (UI/mL)
Anti A
T1R
Abs (
UI/m
L)
r2=0.82P<0.0001
Reactivity to
apoptot
ic cells
by FA
CS
Reactivity to MDA
by EL
ISA
0
2000
4000
6000
U/m
L (
ELIS
A)
or
MF
I (F
AC
S)
AMVR
Stable
P=0.27
P=0.87
AMVR
StableAMVR
Stable
AMVR
StableAMVR
Stable
AMVR
StableAMVR
Stable
E.#
500<MFI<1000
1000<MFI<3000
MFI>3000
Calculated treshold
(MFI)
Positivity among AMVR
cases (n)
A4GALT 3765 0AGRIN 600 3Angiotensinogen 17718 0ARHGDIB 880 0AURKA 10546 2C4B 75 0CHAF1B 19078 0CXCL11 103 0CXCL9 82 0CYCLOPHILIN 2051 0eIF2-α 12023 0ENO-1 14695 0GAD2 13246 0GDNF 396 0HNRNPK 16589 0ICAM-1 496 3IFI16 5314 0IFN-γ 1056 1IL2RA 64 4IL7R 69 0INSULIN 83 0KHSRP 3767 0LAMIN-A 3082 6Lamin-B1 267 0MYOSIN 4335 0NEUROPHILIN-1 182 3NUSAP1 10071 0PA2G4 20210 0PEROXIREDOXIN 8262 2PKC-Z 8260 8PLUNC 12312 0PSMC4 8836 0PTPIA2 8287 0PTPN22 531 0RPL7 6129 0SPDYA 1581 0TNF-α 4217 3Reg3a 8991 0ERBB3 4039 0CD36 4366 0NCL 218 0PECR 16775 0TRIM21 24616 0PSMA4 13648 0F3 8817 0TROVE2 11648 0IFIH1 14543 0TubA1A 18479 0TubA1B 14603 0TubA1C 11857 0TubB 14520 0Perlican 8755 2PRKRIP1 6372 0EDNRA 5038 0FLRT2 14614 2Vimentin 19060 0Myl 3 1487 0
Collagen I 303 2Collagen II 220 1Collagen III 317 2Collagen IV 156 0collagen V 41 1
Stable (n=10) AMVR (n=23)
Delville M, Lamarthee B, …, Anglicheau D. J Am Soc Nephrol. 2019 Apr;30(4):692-709)
0 5 10 15 200
750
1500
2250
3000
Anti-ETAR Abs (UI/mL)
Re
ativity t
o M
DA
by D
EL
FIA
(U
I/m
L)
r2=0.26
P=0.0065
A.# B.#
Anti-ETAR Abs Anti-AT1R Abs
0
5
10
15
20
U/m
L
AMVR
StableP=0.39 P=0.14
C.# D.#
Reactivity to apoptotic cells
by FACS
Reactivity to MDA by
DELFIA
AMVR
StableAMVR
Stable
0 5 10 15 200
5
10
15
20
Anti-ETAR Abs (UI/mL)
Anti A
T1R
Abs (
UI/m
L)
r2=0.82P<0.0001
Reactivity to
apoptot
ic cells
by FA
CS
Reactivity to MDA
by EL
ISA
0
2000
4000
6000
U/m
L (
ELIS
A)
or
MF
I (F
AC
S)
AMVR
Stable
P=0.27
P=0.87
AMVR
StableAMVR
Stable
AMVR
StableAMVR
Stable
AMVR
StableAMVR
Stable
E.#
500<MFI<1000
1000<MFI<3000
MFI>3000
Calculated treshold
(MFI)
Positivity among AMVR
cases (n)
A4GALT 3765 0AGRIN 600 3Angiotensinogen 17718 0ARHGDIB 880 0AURKA 10546 2C4B 75 0CHAF1B 19078 0CXCL11 103 0CXCL9 82 0CYCLOPHILIN 2051 0eIF2-α 12023 0ENO-1 14695 0GAD2 13246 0GDNF 396 0HNRNPK 16589 0ICAM-1 496 3IFI16 5314 0IFN-γ 1056 1IL2RA 64 4IL7R 69 0INSULIN 83 0KHSRP 3767 0LAMIN-A 3082 6Lamin-B1 267 0MYOSIN 4335 0NEUROPHILIN-1 182 3NUSAP1 10071 0PA2G4 20210 0PEROXIREDOXIN 8262 2PKC-Z 8260 8PLUNC 12312 0PSMC4 8836 0PTPIA2 8287 0PTPN22 531 0RPL7 6129 0SPDYA 1581 0TNF-α 4217 3Reg3a 8991 0ERBB3 4039 0CD36 4366 0NCL 218 0PECR 16775 0TRIM21 24616 0PSMA4 13648 0F3 8817 0TROVE2 11648 0IFIH1 14543 0TubA1A 18479 0TubA1B 14603 0TubA1C 11857 0TubB 14520 0Perlican 8755 2PRKRIP1 6372 0EDNRA 5038 0FLRT2 14614 2Vimentin 19060 0Myl 3 1487 0
Collagen I 303 2Collagen II 220 1Collagen III 317 2Collagen IV 156 0collagen V 41 1
Stable (n=10) AMVR (n=23)
Analysis of the seroreactivity of serum samples toward 62 non-HLA antigens using single-antigen flow bead assays
Microvascular endothelial cells are the main targets of AECAs
Kidney International (2006) 69, 1633–1640
(CiGEnC, Conditionally immortalized human Glomerular Endothelial Cells).
Delville M, Lamarthee B, …, Anglicheau D. J Am Soc Nephrol. 2019 Apr;30(4):692-709)
Endothelial cell crossmatch assay
Serumof
AMVR patientswithout
anti-HLA Ab
Pool of sera fromhealthy
volunteers
Detection of non anti-HLA AECAs
The seroreactivity against glomerular ECs is significantly increased in sera from patients with AMVR
Endothelial cell crossmatch assay
HV No AMVR AMVR0
2
4
6
8
10
12
MF
I (F
old
-incre
ase)
P<0.0001
****ns
***
Con
trol
AM
VR#1
AM
VR#2
AM
VR#3
AM
VR#4
AM
VR#5
AM
VR#6
AM
VR#7
AM
VR#8
AM
VR#9
AMVR#1
0
AMVR#1
1
AMVR#1
2
AMVR#1
3
AMVR#1
4
AMVR#1
5
AMVR#1
6
AMVR#1
7
AMVR#1
8
AMVR#1
90
50
100
150
200
250
MF
I (G
eo-M
ean)
!
unstimulatedstimulated
Macrovascular ECs
Con
trol
AM
VR#1
AM
VR#2
AM
VR#3
AM
VR#4
AM
VR#5
AM
VR#6
AM
VR#7
AM
VR#8
AM
VR#9
AM
VR#1
0
AM
VR#1
1
AM
VR#1
2
AM
VR#1
3
AM
VR#1
4
AM
VR#1
5
AM
VR#1
6
AM
VR#1
7
AM
VR#1
8
AM
VR#1
90
50
100
150
200
250
450
500
MF
I (G
eo-M
ean)
!
unstimulatedstimulatedMicrovascular ECs
A.#
B.#
C.#
D.#Sample ID Fold
No AMVR#1 1.20
No AMVR#2 1.21
No AMVR#3 1.24
No AMVR#4 1.33
No AMVR#5 1.41
No AMVR#6 1.48
AMVR#12 1.64
No AMVR#7 1.66
No AMVR#8 1.72
AMVR#7 1.96
AMVR#2 2.02
No AMVR#9 2.04
AMVR#13 2.06
AMVR#4 2.16
AMVR#9 2.18
AMVR#6 2.24
No AMVR#10 2.39
AMVR#17 2.46
AMVR#14 2.54
AMVR#5 2.56
AMVR#3 2.58
AMVR#18 2.73
AMVR#20 2.79
AMVR#10 2.86
AMVR#8 2.86
AMVR#16 2.97
AMVR#15 3.15
AMVR#1 3.22
AMVR#11 5.00
AMVR#19 8.23
Delville M, Lamarthee B, …, Anglicheau D. J Am Soc Nephrol. 2019 Apr;30(4):692-709)
#11
#19
+/-differentiatedCiGEnC
SecondaryIgG
Flowcytometry
AECAs
HV
Detection of non anti-HLA AECAs in AMVR patients serum at day 0 and at rejection
Day 0
AMVR#18
AMVR#20
AMVR#11
IgG binding
AMVR#19
IgG binding
Pooled HV AMVR#11 AMVR#19
2 4 8 16 32 64 128 2560
100
200
300
400
500
dilution (1/x)
MF
I (G
eo-M
ean) HV (AB serum pool)
AMVR#11 at Day 0
AMVR#11 at rejection
A.# B.#
C.#
Undifferentiated glomerular ECs
Differentiated glomerular ECs
Kidney epithelial cells
At rejection
Delville M, Lamarthee B, …, Anglicheau D. J Am Soc Nephrol. 2019 Apr;30(4):692-709)
Endothelial cell crossmatch assay
Detection after 1/128 dilution: High antibody titer
No effect of cell stimulation
Specificity of AMVR seroreactivity against Micro vascular endothelial vs Macro endothelial antigens Assessment of Macro vs Micro endothelial transcriptomes to identify differentially expressed genes
HV No AMVR AMVR0
2
4
6
8
10
12
MF
I (F
old
-incre
ase)
P<0.0001
****ns
***
Con
trol
AM
VR#1
AM
VR#2
AM
VR#3
AM
VR#4
AM
VR#5
AM
VR#6
AM
VR#7
AM
VR#8
AM
VR#9
AMVR#1
0
AMVR#1
1
AMVR#1
2
AMVR#1
3
AMVR#1
4
AMVR#1
5
AMVR#1
6
AMVR#1
7
AMVR#1
8
AMVR#1
90
50
100
150
200
250
MF
I (G
eo-M
ean)
!
unstimulatedstimulated
Macrovascular ECs
Con
trol
AM
VR#1
AM
VR#2
AM
VR#3
AM
VR#4
AM
VR#5
AM
VR#6
AM
VR#7
AM
VR#8
AM
VR#9
AM
VR#1
0
AM
VR#1
1
AM
VR#1
2
AM
VR#1
3
AM
VR#1
4
AM
VR#1
5
AM
VR#1
6
AM
VR#1
7
AM
VR#1
8
AM
VR#1
90
50
100
150
200
250
450
500
MF
I (G
eo-M
ean)
!
unstimulatedstimulatedMicrovascular ECs
A.#
B.#
C.#
D.#Sample ID Fold
No AMVR#1 1.20
No AMVR#2 1.21
No AMVR#3 1.24
No AMVR#4 1.33
No AMVR#5 1.41
No AMVR#6 1.48
AMVR#12 1.64
No AMVR#7 1.66
No AMVR#8 1.72
AMVR#7 1.96
AMVR#2 2.02
No AMVR#9 2.04
AMVR#13 2.06
AMVR#4 2.16
AMVR#9 2.18
AMVR#6 2.24
No AMVR#10 2.39
AMVR#17 2.46
AMVR#14 2.54
AMVR#5 2.56
AMVR#3 2.58
AMVR#18 2.73
AMVR#20 2.79
AMVR#10 2.86
AMVR#8 2.86
AMVR#16 2.97
AMVR#15 3.15
AMVR#1 3.22
AMVR#11 5.00
AMVR#19 8.23
HV No AMVR AMVR0
2
4
6
8
10
12
MF
I (F
old
-incre
ase)
P<0.0001
****ns
***
Con
trol
AM
VR#1
AM
VR#2
AM
VR#3
AM
VR#4
AM
VR#5
AM
VR#6
AM
VR#7
AM
VR#8
AM
VR#9
AM
VR#1
0
AM
VR#1
1
AM
VR#1
2
AM
VR#1
3
AM
VR#1
4
AM
VR#1
5
AM
VR#1
6
AM
VR#1
7
AM
VR#1
8
AM
VR#1
90
50
100
150
200
250
MF
I (G
eo-M
ean)
!
unstimulatedstimulated
Macrovascular ECs
Con
trol
AM
VR#1
AM
VR#2
AM
VR#3
AM
VR#4
AM
VR#5
AM
VR#6
AM
VR#7
AM
VR#8
AM
VR#9
AM
VR#1
0
AM
VR#1
1
AM
VR#1
2
AM
VR#1
3
AM
VR#1
4
AM
VR#1
5
AM
VR#1
6
AM
VR#1
7
AM
VR#1
8
AM
VR#1
90
50
100
150
200
250
450
500
MF
I (G
eo-M
ean)
!
unstimulatedstimulatedMicrovascular ECs
A.#
B.#
C.#
D.#Sample ID Fold
No AMVR#1 1.20
No AMVR#2 1.21
No AMVR#3 1.24
No AMVR#4 1.33
No AMVR#5 1.41
No AMVR#6 1.48
AMVR#12 1.64
No AMVR#7 1.66
No AMVR#8 1.72
AMVR#7 1.96
AMVR#2 2.02
No AMVR#9 2.04
AMVR#13 2.06
AMVR#4 2.16
AMVR#9 2.18
AMVR#6 2.24
No AMVR#10 2.39
AMVR#17 2.46
AMVR#14 2.54
AMVR#5 2.56
AMVR#3 2.58
AMVR#18 2.73
AMVR#20 2.79
AMVR#10 2.86
AMVR#8 2.86
AMVR#16 2.97
AMVR#15 3.15
AMVR#1 3.22
AMVR#11 5.00
AMVR#19 8.23
Delville M, Lamarthee B, …, Anglicheau D. J Am Soc Nephrol. 2019 Apr;30(4):692-709)
Endothelial cell crossmatch assay
Identification of more than 2000 genes significantly overexpressed in microvascular endothelial cells
Transcriptomic profiles of micro and macro ECs
differentially expressed genes in micro vs macro endothelial cells
Overexpressedgenes in
Microvascularendothelium
Delville M, Lamarthee B, …, Anglicheau D. J Am Soc Nephrol. 2019 Apr;30(4):692-709)
Assessment of patients seroreactivity
Delville M, Lamarthee B, …, Anglicheau D. J Am Soc Nephrol. 2019 Apr;30(4):692-709)
the global Ab response at Day-0 was highlyvariable among individuals…
A.
0.0
0.1
0.2
Macro ECs Micro ECs
B.
C.
3
0
-3
0
1.5
AMVR Stable
Proteomic data/Seroreactivity
Transcriptomic data
Matching for overall Score
PC1
PC
2
AMVR
Stable
… but distinguished sera from patients withAMVR from sera from stable patients
A.
0.0
0.1
0.2
Macro ECs Micro ECs
B.
C.
3
0
-3
0
1.5
AMVR Stable
Proteomic data/Seroreactivity
Transcriptomic data
Matching for overall Score
PC1
PC
2
AMVR
Stable
A.
0.0
0.1
0.2
Macro ECs Micro ECs
B.
C.
3
0
-3
0
1.5
AMVR Stable
Proteomic data/Seroreactivity
Transcriptomic data
Matching for overall Score
PC1
PC
2
AMVR
Stable
RNA-Seq Protein arrays
Integrative analysis
Delville M, Lamarthee B, …, Anglicheau D. J Am Soc Nephrol. 2019 Apr;30(4):692-709)
Delville M, Lamarthee B, …, Anglicheau D. J Am Soc Nephrol. 2019 Apr;30(4):692-709)
Integrative analysis
Delville M, Lamarthee B, …, Anglicheau D. J Am Soc Nephrol. 2019 Apr;30(4):692-709)
Integrative analysis
Delville M, Lamarthee B, …, Anglicheau D. J Am Soc Nephrol. 2019 Apr;30(4):692-709)
Integrative analysis
In silico identified targets are expressed at the protein level in microvascular renal endothelial cells.
Expression of HLA molecules
HLA
AB
CH
LA D
R
CiGEnC cells constitutively express HLA Class I molecules CiGEnC cells induce HLA Class II molecules under stimulation
CiGEnC
CRISPR/Cas9 genome editing
CiGEnCΔHLA
HLA Class I deletion by b2 microglobulin knock-out
HLA Class II deletion by CIITA knock-out
HLA Class I
PECAM1
ICAM2
VEGFR2
vWF
VE-cadherin
HLA Class II
( )+ TNF-α+ IFN-γ
PECAM1ICAM2
VEGFR2
vWF
VE-cadherin
HLA Class IIHLA Class I
( )+ TNF-α+ IFN-γ
The expression of HLA molecules may limit the use of the CiGEnC cells to assess the presence of AECA Abs in highly sensitized patients
We developed a CiGEnc cell lacking HLA Class I and II: CiGEnCΔHLA
Lamarthee B, …, Anglicheau D. (in preparation)
Development of CiGEnCDHLA cells
Development of CiGEnCDHLA cells
Lamarthee B, …, Anglicheau D. (in preparation)
B2M
Exon 2 Exon 1
CIITA
Exon 2 Exon 3
Un
mo
difie
d
CiG
En
CC
iGE
nCΔ
HL
A
HL
A A
BC
HLA DR
94.37%
.40%
48.01%
.09%
.31%
.03%
TNF-α/IFN-γ
Stimulation +-
105
104
103
105
104
103
105
104
103
105
104
103
103
103
104
104
103
103
104
104
Sequential deletion of B2M and CIITA by CRISPR/Cas9
CiG
EnCΔ
HLA
Unm
odifie
d
CiG
EnC
- - ++TNF-α/IFN-γ
Stimulation
HLA ABC HLA DR
Longitudinal IgG reactivity to CiGEnCDHLA cells
Lamarthee B, …, Anglicheau D. (in preparation)
Non anti-HLA AECAs assessment
Day 0 1 year0.5
1
2
4
8
16
32
RF
I (G
EO
Mean p
atient/negative c
ontrol)
P<0.0001
Day 0 4 years
Tx
Serum
We assessed the IgG reactivity to CIGEnCDHLA cells in 156 consecutive patients transplanted at Necker Hospital, Paris, France.
3 years2 years1 year
Large variability of IgG reactivity to CIGENCDHLA cells immediately before transplantation
Highly significant increase of IgG reactivity to CIGENCDHLA cells during the first post-transplant year
Summary
A highly selected cohort of 38 patients with acute microvascular rejection (called AMVR) was identified.
Severe phenotype: Early after Tx Severe microvascular inflammation with a mean Banff g+ptc score of 3.9±0.25 Vasculitis (60.5%), interstitial hemorrhages (31.6%), thrombotic microangiopathy (15.8%)
No significant increase of AT1R, ETAR or natural polyreactive Abs (Nabs). anti-ETAR Abs correlated with anti-AT1R Abs (r2=0.82, P<0.0001) and Nabs (r2=0.26,
P=0.0065) supporting the view of a vast auto-immune response.
An homemade endothelial crossmach assay identified a common IgG response specifically directed to constitutively expressed antigens of microvascular glomerular cells
Protein arrays identified 18 antigenic targets that were specifically found in the AMVR patient’s sera, some of them known to be expressed in the human glomerules.
our results suggest that in vitro cell based assays are needed to assess the presence of endothelial cell antibodies with potential deleterious effect after transplantation.
Summary
A highly selected cohort of 38 patients with acute microvascular rejection (called AMVR) was identified.
Severe phenotype: Early after Tx Severe microvascular inflammation with a mean Banff g+ptc score of 3.9±0.25 Vasculitis (60.5%), interstitial hemorrhages (31.6%), thrombotic microangiopathy (15.8%)
No significant increase of AT1R, ETAR or natural polyreactive Abs (Nabs). anti-ETAR Abs correlated with anti-AT1R Abs (r2=0.82, P<0.0001) and Nabs (r2=0.26,
P=0.0065) supporting the view of a vast auto-immune response.
An homemade endothelial crossmach assay identified a common IgG response specifically directed to constitutively expressed antigens of microvascular glomerular cells
Protein arrays identified 18 antigenic targets that were specifically found in the AMVR patient’s sera, some of them known to be expressed in the human glomerules.
our results suggest that in vitro cell based assays are needed to assess the presence of endothelial cell antibodies with potential deleterious effect after transplantation.
Summary
A highly selected cohort of 38 patients with acute microvascular rejection (called AMVR) was identified.
Severe phenotype: Early after Tx Severe microvascular inflammation with a mean Banff g+ptc score of 3.9±0.25 Vasculitis (60.5%), interstitial hemorrhages (31.6%), thrombotic microangiopathy (15.8%)
No significant increase of AT1R, ETAR or natural polyreactive Abs (Nabs). anti-ETAR Abs correlated with anti-AT1R Abs (r2=0.82, P<0.0001) and Nabs (r2=0.26,
P=0.0065) supporting the view of a vast auto-immune response.
An homemade endothelial crossmach assay identified a common IgG response specifically directed to constitutively expressed antigens of microvascular glomerular cells
Protein arrays identified 18 antigenic targets that were specifically found in the AMVR patient’s sera, some of them known to be expressed in the human glomerules.
our results suggest that in vitro cell based assays are needed to assess the presence of endothelial cell antibodies with potential deleterious effect after transplantation.
Summary
A highly selected cohort of 38 patients with acute microvascular rejection (called AMVR) was identified.
Severe phenotype: Early after Tx Severe microvascular inflammation with a mean Banff g+ptc score of 3.9±0.25 Vasculitis (60.5%), interstitial hemorrhages (31.6%), thrombotic microangiopathy (15.8%)
No significant increase of AT1R, ETAR or natural polyreactive Abs (Nabs). anti-ETAR Abs correlated with anti-AT1R Abs (r2=0.82, P<0.0001) and Nabs (r2=0.26,
P=0.0065) supporting the view of a vast auto-immune response.
A new endothelial crossmach assay identified a common IgG response specifically directed to constitutively expressed antigens of microvascular glomerular cells
Protein arrays identified 18 antigenic targets that were specifically found in the AMVR patient’s sera, some of them known to be expressed in the human glomerules.
our results suggest that in vitro cell based assays are needed to assess the presence of endothelial cell antibodies with potential deleterious effect after transplantation.
Summary
A highly selected cohort of 38 patients with acute microvascular rejection (called AMVR) was identified.
Severe phenotype: Early after Tx Severe microvascular inflammation with a mean Banff g+ptc score of 3.9±0.25 Vasculitis (60.5%), interstitial hemorrhages (31.6%), thrombotic microangiopathy (15.8%)
No significant increase of AT1R, ETAR or natural polyreactive Abs (Nabs). anti-ETAR Abs correlated with anti-AT1R Abs (r2=0.82, P<0.0001) and Nabs (r2=0.26,
P=0.0065) supporting the view of a vast auto-immune response.
A new endothelial crossmach assay identified a common IgG response specifically directed to constitutively expressed antigens of microvascular glomerular cells
Protein arrays identified antigenic targets that were specifically found in the AMVR patient’s sera, some of them known to be expressed in the human glomerulus.
our results suggest that in vitro cell based assays are needed to assess the presence of endothelial cell antibodies with potential deleterious effect after transplantation.
Summary
A highly selected cohort of 38 patients with acute microvascular rejection (called AMVR) was identified.
Severe phenotype: Early after Tx Severe microvascular inflammation with a mean Banff g+ptc score of 3.9±0.25 Vasculitis (60.5%), interstitial hemorrhages (31.6%), thrombotic microangiopathy (15.8%)
No significant increase of AT1R, ETAR or natural polyreactive Abs (Nabs). anti-ETAR Abs correlated with anti-AT1R Abs (r2=0.82, P<0.0001) and Nabs (r2=0.26,
P=0.0065) supporting the view of a vast auto-immune response.
A new endothelial crossmach assay identified a common IgG response specifically directed to constitutively expressed antigens of microvascular glomerular cells
Protein arrays identified antigenic targets that were specifically found in the AMVR patient’s sera, some of them known to be expressed in the human glomerulus.
our results suggest the value of in vitro cell based assays to assess the presence of endothelial cell antibodies with potential deleterious effect after transplantation.
Conclusion: Many challenges !
AECAs represent an heterogeneous group of antibodies
They might target non HLA allo-antigens or tissue specific auto-antigens
Because autoantibodies arise in the course of tissue injury and chronic disease, their role in disease pathogenesis is difficult to prove.
Lack of knowledge of the antigenic specificity of most of these non-HLA targets
Uncertainty on treatment regimens
Possible cross talk between the allo-immune responses and auto immunity: The presence of pre-transplant non-HLA antibodies may increase allo immunity and
allograft rejection Tissue injuries and remodeling resulting from allo-immune responses to anti-HLA, may
potentiate exposure to cryptic self antigens and lead to auto-immunity
Need validated assays for detection of non-HLA Ab to understand clinical relevance.
Large multicentric studies adjusted on anti HLA sensitization and on recent cohorts of patients are still needed.
Delville M, Anglicheau D. Hum Immunol. 2016Jackson AM, Anglicheau D. Human Immunol 2019
Sarma NJ et al, Human Immunol, 2012; Angaswamy N et al, Human Immunol 2013Tait BD et al. Trsplantation 2013; Halloran P. Nature rev nephrol 2014
Acknowledgments
Beatrice CharreauSylvain PagiéAnne Cesbron
Our French and Belgian colleagues
Dr Arzouk, CHU La Pitiè-Salpétrière, ParisDr Bertrand, CHU de RouenPr Caillard, CHU de StrasbourgPr Ducloux, CHU de BesançonDr Garrouste, CHU de Clermont-FerrandDr Gatault, CHU de ToursPr Giral, CHU de NantesPr Hazzan, CHU de LillePr Hertig, CHU Tenon, ParisPr Kamar, CHU de ToulouseDr Ladrière, CHU de NancyPr Le Moine, CHU de BruxellesPr Mariat, CHU de St EtienneDr Matignon, CHU Henri MondorPr Merville, CHU de BordeauxDr Rivalan, CHU de RennesDr Sayag, CHU d’AngersDr Thaunat et Dr Sicard, CHU de LyonDr Vuiblet, CHU de ReimsDr Westeel, CHU d’Amiens
Marianne DelvilleBaptiste LamartheeCarole BurgerMarion RabantJean Paul DuongAlexandre LoupyOlivier AubertNicolas CagnardChristine Bole
Emmanuel ZornSarah See
Jean-Luc Taupin