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

............................ ........................................................

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

600

800

Biopsie

Ñ

Ñ Solumedrol

ÑÑ

Ä

Ä Rituximab

Ä


ÑÑÑ ÑÑ

............................ ........................................................


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


ÑÑÑ ÑÑ

............................ ........................................................


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


ÑÑÑ ÑÑ

............................ ........................................................


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


ÑÑÑ ÑÑ

............................ ........................................................


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


ÑÑÑ ÑÑ

............................ ........................................................


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.



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.



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)



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


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


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 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)


0 5 10 15 200

750

1500

2250

3000


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 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).


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


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


#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



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)

!


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)

!


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)

!


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)

!


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



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


Assessment of patients seroreactivity


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


Transcriptomic data


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


Transcriptomic data


PC1

PC

2

AMVR

Stable

RNA-Seq Protein arrays

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


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


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 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.


Summary






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.


Summary






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

non-hla anti-endothelial cell antibodies: an old problem ... · non-hla anti-endothelial cell...

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