nÉogenÈse lymphoÏde au cours du rejet chronique: … · place in the other arterial layers. ......

NÉOGENÈSE LYMPHOÏDE AU COURS DU REJET CHRONIQUE:

UNE REPONSE HUMORALE ALLOIMMUNE LOCALE?

Olivier ThaunatEdouard Herriot Hospital

INSERM U851/Centaure NetworkLyon, France

[email protected]

Chronic allograft dysfunction

www.ctstransplant.org/

Renal graft survival according to the date of transplantation

Chronic rejection

chronic rejection

Pascual, NEJM, 2002

Hillebrands, Exp. Rev. Mol. Med, 2003

Chronic rejection

Aortic interposition(or carotid)

donor recipient

Schmitz-Rixen, J Vasc Surg, 1988Mennander, Arterioscler Thromb, 1991Plissonnier, Arterioscler Thromb, 1991

Experimental model of chronic rejection

Histoincompatible strains

Aortic interposition(or carotid)

donor recipient

Schmitz-Rixen, J Vasc Surg, 1988Mennander, Arterioscler Thromb, 1991Plissonnier, Arterioscler Thromb, 1991

Experimental model of chronic rejection

Histoincompatible strains

*Surgically feasible*Lesions develop within 3 months *Lesions are similar to the clinic*No antithrombotic agent*No immunosuppression

Chronic vascular rejection

Normal artery Chronically rejected artery

Adventitial infiltrate may play a critical role in the development of chronic vascular rejection lesions

Disproportion between the adventitial inflammation and the low number of adventitial allogenic targets

Chronologically related to the events taking place in the other arterial layers

Aortic Interposition

BN (RT1An)

donor

LEW (RT1Al)

recipient

ImmunohistologyElectron microscopy

Organoculture

Experimental protocol

d10 M1M2

Microdissection

Flow cytometry

Adventitial angiogenesis during chronic vascular rejection

RECA

Aortic grafts1 month post transplantation

Isograft Allograft

Chronic rejection is associated with an intense angiogenesis in the adventitia

Endothelial cells of adventitial vasa vasorum acquires HEV phenotype during rejection

Isograft Allograft

Aortic grafts10 days post transplantation

Adventitial angiogenesis during chronic vascular rejection

Adventitial inflammatory infiltrate

Media

Adventitia

NeointimaLumen

T Lymphocytes (R73)

Aortic grafts1 month post transplantation

Adventitial T lymphocytes: *represent the main population infiltrating the graft during CR*exhibit a diffuse pattern of infiltration

Adventitial T cell infiltrate

Over a period of 1 month, adventitial T-cell infiltrate shifted from an initial cytotoxic to a B-cell helper phenotype

CD4+ CD8+ CD4+ CD8+ CD4+ CD8+

***

***

**

* *

Adventitial B cell infiltrate

B Lymphocytes

Centre germinatif Prolifération ApoptoseApoptosisProliferationGerminal centers

Ontogeny of B lymphocytes

B

Lymph node

Germinal center

HEV

Bone marrowC

ircul

atio

n


B

Lymph node

Germinal center

HEV

Bone marrowC

ircul

atio

n

BCirc

u


B

Lymph node

Germinal center

HEV

Bone marrowC

ircul

atio

n

BCirc

uTh

B

DC


B

Lymph node

Germinal center

HEV

Bone marrowC

ircul

atio

n

BCirc

uTh

B

DC

BAID


B

Lymph node

Germinal center

HEV

Bone marrowC

ircul

atio

n

BCirc

uTh

B

DC

BAID

FDC

FDC

FDC B BB

B

B

B

Somatic hypermutationsClass switch


B

Lymph node

Germinal center

HEV

Bone marrowC

ircul

atio

n

BCirc

uTh

B

DC

BAID

FDC

FDC

FDC B BB

B

B

B

Somatic hypermutationsClass switch

B

Memory B

Plasmacells

B

Hypothesis

Are these nodular lymphoid structures ectopic germinal centers?

Are these ectopic germinal centers involved in the rejection process?


Aortic graft

Spleen

Draininglymph node

Rat aortic interpositionmodel


Aortic graft

Spleen

Draininglymph node


Ab purification from culture supernatants

Organoculturesp


Aortic graft

Spleen

Draininglymph node



Organoculturesp

Incubation

Recipient fibroblasts

Recipient fibroblasts Transfected with donor’s MHC


Aortic graft

Spleen

Draininglymph node



Organoculturesp

Incubation

Recipient fibroblasts

Recipient fibroblasts Transfected with donor’s MHC

Flow cytometry

Intragraft humoral alloimmune response

Thaunat, PNAS, 2005

Adventitia of chronically rejected aortic grafts is a site of production of alloantibodies

Lymphoid neogenesis in chronic rejection

The progressive organization of chronic inflammatory infiltrate into

functional ectopic germinal centers (tertiary lymphoid organs; TLOs) have

been named lymphoid neogenesis.Schroder, PNAS, 1996Kratz, J Exp Med, 1996

Lymphoid neogenesis takes place during experimental chronic

rejectionThaunat, PNAS, 2005

Badoura, Am J Transplant, 2005

=> Chronically rejected graft is at the same time the target and a site of

production of alloantibodies

What is the validity of this experimental findings in the clinical settings

?

Human grafts explanted for chronic rejection

Heart Kidney

HES x 5Tertiary lymphoid organs develop within the graft during clinical chronic

rejectionThaunat, Curr Opin Immunol, 2006

Microarchitecture of TLOs

Intragraft TLOs display the same microarchitecture as “professional” secondary lymphoid organs

HES HEV Lymphatics

DC Lamp CD3 FDC

Germinal center reaction

ThDC

B

Priming

IgD

Naive B cell

B

IgM

Germinal center

BAID

IgGIgAIgMIgE

Isotype switching Somatic hypermutation

IgD

Functionality of intragraft TLOs

Expression of AID and Ig class switch in intragraft TLOs

IgDAID

Immunohistochemistry X10

Are lymphoid neogenesis features only evidenced in explanted grafts(i.e. terminally rejected grafts)

?

Lymphoid neogenesis features on graft biopsy

Mr AL..; 28 years old; initial nephropathy = FSG

Renal transplantation 5 years ago

Slow increase of creatininemia (200 µmol/l vs 130)

=> biopsy

Trichrome CD20

What are the mechanisms triggering the organization of inflammatory infiltrates into

TLOs during chronic rejection?

Mebius, Nat Rev Immunol, 2003

Organogenesis of professional lymphoid tissues

Hypothesis: is lymphoid neogenesis the recapitulation of lymphoid organogenesis?

Design of the study

20 samples

- Renal grafts removed for terminal failure due to chronic rejection

12 Controls

- 3 renal grafts removed for primary failure< 10 days post-transplantation(1 arterial and 2 venous thrombosis)

- 3 renal grafts removed for non-immune mediated late failure (1 angiosarcoma, 2 recurrence of FSG)

- 6 native kidneys removed for carcinoma

Prospective collection of explanted kidney allografts (over 4 years in 5 French transplantation centers)

Q-PCR analysis of the level of expressionof LO genes in these tissues

C4

C3

C2

C1 controls

samples

Rel

ativ

e ge

ne e

xpre

ssio

n (Q

PC

R)

Relative expression of the genes involved in LO in chronically rejected grafts

CR allografts display heterogeneous levels of expression of the genes involved in LO

CR is associated with the expression of a set of genes involved in LO

LO genes

Experimental protocolExplanted graft


Cell suspension*detection of plasmacells

ELISPOT



ELISPOT

Organocultures



ELISPOT

Organocultures

* quantification of IgG subclassesELISA

Supernatants

High level of expression of the genes involved in LO correlates with a high number of infiltrating plasmacells =>

intense local production of IgG

C4C3C2C1

C4C3C2C1

ELISA(IgG subclass)

ELISPOT IgG(Plasmacells)

Plasmacells & IgG production according to the level of expression of LO genes


Organocultures

* Alloantibody detection &identificationLuminex assay

Explanted graft

Supernatants

7.7±0.39.6±8.681.8±31.6%C4

0.7±0.10.9±0.232.3±31.6%C3

0.6±0.21.1±0.224.1±13%C2

--0%C1

IntensityMFI max /

MFI control+

Diversitynb of specificity /nb of mismatch

Density% of positive wells

Characteristics of intragraft humoral alloimmune response according to the level

of expression of LO genes

High level of expression of LO genes correlates with a dense, diverse and intense local humoral response

against the graft

1) Inflammatory infiltrates organizes themselves into functionalectopic germinal centers (lymphoid neogenesis) within chronically rejected grafts

2) Lymphoid neogenesis seems to recapitulate the genetic program triggered during the organogenesis of secondary lymphoid organs in the embryo

3) Lymphoid neogenesis results in the development of a local alloimmune response that likely participates in the chronic destruction process

Lymphoid neogenesis in clinical chronic rejection

Is lymphoid neogenesis a therapeutic target for chronic rejection?

Remaining questions

1) Is Lymphoid neogenesis a therapeutic target for chronic rejection?

2) (If so ) What would be the best therapeutic strategy to traget this local immune response?

Impact of Rtx therapy on intragraft lymphoid neogenesis

3.8 months10.3 monthsTime from the last course of Rtx

3/mm35/mm3CD19 count in periphery

12Number of RTX courses

14,5 years2 yearsTime from transplantation

Patient #2Patient #1(At the time of the explantation)

We obtained 2 grafts explanted from patients on RTX therapy initiated for C4d positive rejection refractory to IVIG and plasmapheresis

Satisfactory CD19 depletion in the periphery was not predictive of a complete intragraft B cell-depletion

Flow cytometry analysis of the composition of intragraft inflammatory infiltrate after RTX

Patient #1 Patient #2

Immunohistological analysis of the microarchitecture of intragraft inflammatory

infiltrate after RTXPatient #1 Patient #2

RTX was insufficient to disrupt the spatial organizationof intragraft TLOs

CD20

Organocultures

* Alloantibody detection &identificationLuminex assay

Explanted graftAfter RTX

Supernatants

Evaluation of the functionality of TLOs after RTX therapy

Evaluation of the functionality of TLOs after RTX therapy

Persistence of intragraft TLOs after RTX correlated with a persistent local alloAb synthesis

Impact of Rtx therapy on intragraft lymphoid neogenesis

1) Satisfactory CD19 depletion in the periphery was not predictive of a total intragraft depletion

2) Persistence of intragraft B cell correlated with a local alloAb synthesis

=> Why intragraft B cells escape rituximab-induced depletion?

Inflammatory cells provide intragraft B cells with BAFF survival signal

Inflammatory microenvironment rescues B cells from rituximab therapy

BAFF

Q-PCR immunohistochemistry

AcknowledgementsINSERM U872Antonino NicolettiStéphanie GraffMarie-Caroline Dieu-Nosjean

INSERM U698Jean-Baptiste MichelLiliane LouedecJiangping Dai

Nephrology, Urology, andPathology Departments of:

Necker (Paris)Henri Mondor (Créteil)Foch (Suresnes)Pasteur (Nice)Edouard Herriot (Lyon)

Emmanuel MorelonNatacha PateyChantal GautreauSophie LechatonCassuto-Viguier Elisabeth

nÉogenÈse lymphoÏde au cours du rejet chronique: … · place in the other arterial layers. ......

Documents