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NÉOGENÈSE LYMPHOÏDE AU COURS DU REJET CHRONIQUE:
UNE REPONSE HUMORALE ALLOIMMUNE LOCALE?
Olivier ThaunatEdouard Herriot Hospital
INSERM U851/Centaure NetworkLyon, France
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Chronic allograft dysfunction
www.ctstransplant.org/
Renal graft survival according to the date of transplantation
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Chronic allograft dysfunction
www.ctstransplant.org/
Renal graft survival according to the date of transplantation
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Chronic allograft dysfunction
www.ctstransplant.org/
Renal graft survival according to the date of transplantation
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Chronic rejection
chronic rejection
Pascual, NEJM, 2002
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Hillebrands, Exp. Rev. Mol. Med, 2003
Chronic rejection
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Hillebrands, Exp. Rev. Mol. Med, 2003
Chronic rejection
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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
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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
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Chronic vascular rejection
Normal artery Chronically rejected artery
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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
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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
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Aortic Interposition
BN (RT1An)
donor
LEW (RT1Al)
recipient
ImmunohistologyElectron microscopy
Organoculture
Experimental protocol
d10 M1M2
Microdissection
Flow cytometry
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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
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Endothelial cells of adventitial vasa vasorum acquires HEV phenotype during rejection
Isograft Allograft
Aortic grafts10 days post transplantation
Adventitial angiogenesis during chronic vascular rejection
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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
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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+
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* *
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Adventitial B cell infiltrate
B Lymphocytes
Centre germinatif Prolifération ApoptoseApoptosisProliferationGerminal centers
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Ontogeny of B lymphocytes
B
Lymph node
Germinal center
HEV
Bone marrowC
ircul
atio
n
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Ontogeny of B lymphocytes
B
Lymph node
Germinal center
HEV
Bone marrowC
ircul
atio
n
BCirc
u
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Ontogeny of B lymphocytes
B
Lymph node
Germinal center
HEV
Bone marrowC
ircul
atio
n
BCirc
uTh
B
DC
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Ontogeny of B lymphocytes
B
Lymph node
Germinal center
HEV
Bone marrowC
ircul
atio
n
BCirc
uTh
B
DC
BAID
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Ontogeny of B lymphocytes
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
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Ontogeny of B lymphocytes
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
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Hypothesis
Are these nodular lymphoid structures ectopic germinal centers?
Are these ectopic germinal centers involved in the rejection process?
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Experimental protocol
Aortic graft
Spleen
Draininglymph node
Rat aortic interpositionmodel
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Experimental protocol
Aortic graft
Spleen
Draininglymph node
Rat aortic interpositionmodel
Ab purification from culture supernatants
Organoculturesp
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Experimental protocol
Aortic graft
Spleen
Draininglymph node
Rat aortic interpositionmodel
Ab purification from culture supernatants
Organoculturesp
Incubation
Recipient fibroblasts
Recipient fibroblasts Transfected with donor’s MHC
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Experimental protocol
Aortic graft
Spleen
Draininglymph node
Rat aortic interpositionmodel
Ab purification from culture supernatants
Organoculturesp
Incubation
Recipient fibroblasts
Recipient fibroblasts Transfected with donor’s MHC
Flow cytometry
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Intragraft humoral alloimmune response
Thaunat, PNAS, 2005
Adventitia of chronically rejected aortic grafts is a site of production of alloantibodies
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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
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What is the validity of this experimental findings in the clinical settings
?
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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
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Microarchitecture of TLOs
Intragraft TLOs display the same microarchitecture as “professional” secondary lymphoid organs
HES HEV Lymphatics
DC Lamp CD3 FDC
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Germinal center reaction
ThDC
B
Priming
IgD
Naive B cell
B
IgM
Germinal center
BAID
IgGIgAIgMIgE
Isotype switching Somatic hypermutation
IgD
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Functionality of intragraft TLOs
Expression of AID and Ig class switch in intragraft TLOs
IgDAID
Immunohistochemistry X10
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Are lymphoid neogenesis features only evidenced in explanted grafts(i.e. terminally rejected grafts)
?
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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
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What are the mechanisms triggering the organization of inflammatory infiltrates into
TLOs during chronic rejection?
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Mebius, Nat Rev Immunol, 2003
Organogenesis of professional lymphoid tissues
Hypothesis: is lymphoid neogenesis the recapitulation of lymphoid organogenesis?
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Mebius, Nat Rev Immunol, 2003
Organogenesis of professional lymphoid tissues
Hypothesis: is lymphoid neogenesis the recapitulation of lymphoid organogenesis?
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Mebius, Nat Rev Immunol, 2003
Organogenesis of professional lymphoid tissues
Hypothesis: is lymphoid neogenesis the recapitulation of lymphoid organogenesis?
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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
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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
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Experimental protocolExplanted graft
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Experimental protocolExplanted graft
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Experimental protocolExplanted graft
Cell suspension*detection of plasmacells
ELISPOT
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Experimental protocolExplanted graft
Cell suspension*detection of plasmacells
ELISPOT
Organocultures
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Experimental protocolExplanted graft
Cell suspension*detection of plasmacells
ELISPOT
Organocultures
* quantification of IgG subclassesELISA
Supernatants
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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
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Experimental protocol
Organocultures
* Alloantibody detection &identificationLuminex assay
Explanted graft
Supernatants
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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
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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?
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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?
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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
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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
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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
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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
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Organocultures
* Alloantibody detection &identificationLuminex assay
Explanted graftAfter RTX
Supernatants
Evaluation of the functionality of TLOs after RTX therapy
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Evaluation of the functionality of TLOs after RTX therapy
Persistence of intragraft TLOs after RTX correlated with a persistent local alloAb synthesis
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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?
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Inflammatory cells provide intragraft B cells with BAFF survival signal
Inflammatory microenvironment rescues B cells from rituximab therapy
BAFF
Q-PCR immunohistochemistry
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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