Aplasie médullaire Physiopathologie -...

Aplasie médullaire Physiopathologie

Gérard SociéHôpital St Louis / Université Paris VII

Remerciements:- NS Young- A Risitano- J Maciewsky


Les 2 problèmes !moelle vide maladie rare

Takaku et al, Blood 2010

AA

Moelle osseuse

Thymus

Tiss

u pé

riph

ériq

ue

Cellules souches

ProgéniteurMyélo-érythroïde

CFU-GEMM

Progéniteurlymphoïde

Pro-B

Pro-NK

Pro-T

Pré-B Lymphocyte BPlasmocyte

NK

Cellule dendritique

Pré-T

T auxiliaire

T cytotoxique

Compartiment des cellules souches

Compartiment des progéniteurs

Compartiment de maturation Précurseurs et cellules matures

sang

Cellules dendritiques

CFU-GM

CFU-M Pro-Monocyte Monocyte

Macrophage

CFU-G neutrophile neutrophile

éosinophileCFU-Eo éosinophile

basophileCFU-Baso basophileMastocyte

BFU-MK

BFU-E

Mégacaryocyte Plaquettes

Érythroblaste Érythrocyte

Myé

locy

tes

Gra

nulo

cyte

s

ProgéniteurLympho-myéloïde


CSH humaines

CSH

SRC

Cultures à long terme

Prog

énit

eurs

LTC-ICMy, Ly

Tests clonogéniques

CFC

Cellu

les

en c

ours

de m

atur

atio

n

Caractéristiques phénotypiques



Références Nbre patients Résultats

Marsh et al.1990 32

Bacigalupo et al.1992 46

Stark et al.1993 17

Diminution des CFC n=31

Diminution des CFC n= 46

Diminution des CFC n=15

Progéniteurs déterminés


Progéniteurs primitifs

Moelle totale; LTC-IC

CD34


Maciejewski, Blood, 1996

Nombre /105 CMN MoelleCD34+ CFC LTC-IC LTC-IC /CD34+

Control 2890+200 210+120 1,95+0,5 1/1280

AA modérée 854+400 27+10 0,29+0,13 1/1220

AA sévère 230+50 1,5+1 0,19+0,03 1/1242

Diminution des LTC-IC corrélée avec la diminution des cellules CD34+




Maciejewski, Blood, 1996

Clonogénicité Nombre CFC/ 1 LTC-IC des CD34+purifiées

Control 1/13 (8-20%) 4,3+1

AA modérée 1/60 (1-2%) 2+0,4

AA sévère 1/133 (1%) 1,27+0,1

Capacité de prolifération des CD34+ et des LTC-IC diminués



Stroma irradié Mo normale

CD34+ Mo d’AA CD34+ Mo normale

Stroma irradié Mo d’AA

CD34+ Mo normale

Stroma irradié Mo normale

P<0,001

2 3 4 5 6

1 000

100

10

Nom

bre

CFU

-GM

/ p

uits

Normal

CD34 Nl/stroAA

CD34 AA/stro Nl

Marsh, BJH, 1996Semaines

Aplasie médullaire PhysiopathologieProgéniteurs primitifs

Maciejewski JP, et al. Br J Haematol. 1995; 91:245–52.

Control

SAA

CD95 (Fas) FITC

CD34

PE

% o

f Fas

Exp

ress

ion

wit

hin

CD34

+ ce

lls

NC AA recAA MDS HC



Over-expressed

• Apoptosis

• Stress response

• Cytokine/chemokine transduction

• Defense/immune response genes

• Cell cycle/proliferation inhibitors

Down-expressed

• Cell cycle/proliferation promoters

CD34

“…the transcriptome analysis of HSC in AA is consistent with the presence of stressed, immunologically activated or dying target cells rather than of an intrinsically abnormal population.“

“…the transcriptome analysis of HSC in AA is consistent with the presence of stressed, immunologically activated or dying target cells rather than of an intrinsically abnormal population.“


Maladie dysimunitaire

Clinique Reconstitution autologue après allogreffe de moelle Traitement de référence = Immunosuppresseurs

Biologie Lymphocytes T activés (sang et moelle) Phénotype Th1 Action directe cytotoxique ou indirecte (IFN, TNF, MIP1 )

Young, ASH, 2012

Aplasie médullaire PhysiopathologieCIRCULATING ACTIVATED SUPPRESSOR T LYMPHOCYTES IN APLASTIC ANEMIAN.C. Zoumbos, P. Gascon, J.Y. Djeu, S.R. Trost, and N.S. YoungVolume 312 January 31, 1985 Number 5

Perf

orin

PE

Gra

nzym

e B

PE

CD8 PECY5 CD8 PECY5

Aplasie médullaire PhysiopathologieAnti-IFN mAb may restore impaired growth of hematopoietic progenitors in vitro

γ-IFN and TNF-α induce apoptosis of hematopoietic progenitors in vitro

Bone marrow stromal cells transduced with γ-IFN impair hematopoiesis by inducing apoptosis of hematopoietic progenitors


Normal control

SAA patient



Young, N. S. et al. Blood 2006;108:2509-2519


T-bet•T-box family transcription factor essential for Th1 development

•acts downstream Itk and PKC (mostly PKC-Θ)

T-bet


Treg Solomou et al., Blood 2007

Peffault de Latour et al., Blood 2010Th17

•Increased in AA patients

•Correlate with disease status

•Normalize after treatment in good responders only


Tregs Th1 Th2 Th17Severe vs. non-severe AA

Decreased but not statistically different in severe AA

Tregs Th1 Th2 Th17Aplastic anemia vs. controls


Different Tregs (CD25high, Foxp3+) subsets

Impaired in vitro suppressive function of Tregs from AA patients (both in autologous and allogeneic settings)

I

II

III

I; Resting TregsII; Activated TregsIII; Cytokine + / non-Tregs




Représentation anormale de lymphocytes T CD4 à distribution clonale

Zeng, JCI, 2001


Zeng, JCI, 2001

Cytotoxicité(CD34+)

Restreint par HLA-DR

Inhibition des Progéniteurs déterminés


Vb subfamilies

V-CDR3 cDNA pool

Flow cytometry

RT-PCR amplification

CDR3 size analysis

insertion site

Cloning vector

cloning CDR3 sequencing

CASSRFGGYE… …CASSRFGGYE…CARYGANQY..…CASRRPYSEN…CATRGSVNQY...CASYDTNPQH…

TCR-V5.1 FITC TCR-V5.1 FITC

normal skewed

CD28 low

CD28

PE


BMMNCCD33+CD34+


TCR diversity and clonality (on CD4+ subsets)

DiversityDominance

ClonalPolyclonal/oligoclonal


Haematologica | 2013; 98(9)


1. A pathogenic antibody-mediated autoimmune response?

2. Non-pathogenic antibodies as markers of the underlying immune derangement?

3. An Ag-specific B-cell response interplaying with a T-cell response?

These putative auto-Ag may trigger (as whole proteins or derived epitopes) a cytotoxic T-cell response in vitro (but Ag-specific T-cells were never demonstrated in vivo in AA patients)

T-cell poolEscape from self-tolerance maintenance mechanisms

Newly expressed Ags (exogenous, mutated, cross-reacting, tissue specific)

oligoclonal T-cell response

TCR

hematopoietic stem cells

mature blood cells

Immune pathophysiology of aplastic anemia

T-cell poolEscape from self-tolerance maintenance mechanisms

Newly expressed Ags (exogenous, mutated, cross-reacting, tissue specific)

effector mechanisms— Fas— IFN-g

— TNF-a— Perforin mature blood cellsoligoclonal T-cell response

TCR

hematopoietic stem cells

Immune pathophysiology of aplastic anemia


HEMATOLOGY/HEMATOPOIESIS IN“NORMAL” FAMILY MEMBERS WITH TERC MUTATIONS

Hematology

normal peripheral blood counts

mild anemia with macrocytosis

mild thrombocytopenia

Hematopoiesisseverely hypoplastic↓CD34 number↓colony formation↑erythropoietin, thrombopoietin

proband affected sister affected niece unaffected brother


0

2

4

6

8

10

12

14

16

0 20 40 60 80 100

controls

age, years

telo

mer

e le

ngth

, kb

His 412 Tyr

Val 694 MetAla 202 Thr

Cys 772 Tyr

Val 1090 Met

patients

TELOMERE LENGTH IN TERT MUTATION LEUCOCYTES


C204G

TERTA202TH412YV694MY772C

Dyskerin

Pseudoknotdomain

5’5’

CR7 domain

Template

Box

H/A

C A

dom

ain

CR4-

CR5

dom

ain

TERC

G143AA117C

C116T

110-113

GC107-108AG

C72G130 kD

96-97

378-451

C408G

1-316

G305A

G322A

NHP222 kD

NOP1010 kD

GAR125 kD

L37L37

L321VL321V

57 kD

A353VA353V

A2VA2V

T66AT66A

P40RP40RF36VF36V

E41KE41K

L72YL72Y

M350T/IM350T/IG402EG402E

R65TR65T

K39EK39E

TERC

TELOMERASE COMPLEX GENE MUTATIONS AND BONE MARROW FAILURE

TELOMERE DISEASES

EtOH

smoking

immune, virus ,toxin

envi

ronm

ent

LiverLung

BM

genetic penetrance

physiologic aging

Skin/mucosa

DKC Complex Disease Risk Factors

cirrhosis

pulmonary fibrosis

marrow failure

TERT, TERC+ /-

Calado RT, Young NS, NEJM 2009; 361:2353


J. Exp. Med. 2013Vol. 210 No. 7 1311-1329


J. Exp. Med. 2013; Vol. 210 No. 7 1311-1329

Toxic agentsViruses

TPO-RDNA damage

Effector T-cells

Apoptosis

Cycling blockade

Proliferation and survival

Cell cyclingGF-Rs

Growth factors

SBDS

c-mpl

?

Perforine/granzyme

Immune polymorphisms?

Autoantigen, altered protein

TCR

Fas-LFas-R Self-

renewal

DNA repair

Telomerase complex genes

FA genes

Lysis

HLA

Mechanisms of hematopoietic stem cell damage

Risitano AM, et al. Curr Stem Cell Res Ther. 2007;2:39–52.

IFN-γ Reactive oxygen species

NO

TRAIL-Rs

TNF-R

IFN-R

TRAIL

TNF-α

+ inflammatory cytokines

ONSET

immune response

incitingevent

Time

RECOVERY

responses, complete/partial

LATE DISEASE

abnormal clone

relapse of pancytopenia

MDS, AML

Hematopoietic stem cells

Peripheral blood counts

clinical presentation

ACQUIRED APLASTIC ANEMIAN

umbe

r

Aplasie médullaire PhysiopathologieJerez A et al. Blood 2013;122:2453-2459


ARRAY-BASED KARYOTYPING

Chr 7 Chr X

Loss of heterozygozity

Chromosome 7

D7S1842

1

0.48D7S2471

Copy number

1

0.5RQ

CD3+ Bone marrow

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