immune regulation defect in human myasthenia gravis
DESCRIPTION
Immune regulation defect in human Myasthenia Gravis. Sonia Berrih-Aknin CNRS UMR-8162, IPSC. International Conference on Myasthenia Gravis, December 1-2, Paris. Scanner sections of human thymus. ( Meraouna et al, Blood, 2006). Fe male 22 yrs. Fem ale 24 yrs. Fem ale 26 yrs. Control. - PowerPoint PPT PresentationTRANSCRIPT
Immune regulation defect in human Myasthenia Gravis
Sonia Berrih-Aknin
CNRS UMR-8162, IPSC
International Conference on Myasthenia Gravis, December 1-2, Paris
Control MG Cortico+MG Cortico-
Female 26 yrsFemale 22 yrs Female 24 yrs
In Green: follicular dendritic and B cells stained with anti-CD21 antibodies;
in Red; staining with anti-keratin antibodies
Scanner sections of human thymusScanner sections of human thymus(Meraouna et al, Blood, 2006)
Anti-acetylcholine receptor autoimmune response Anti-acetylcholine receptor autoimmune response occurs in the thymus of MG patientsoccurs in the thymus of MG patients
Cytokines
Th2 B
IL-4
T cellsV5.1Fas
resting T
activated T
MHC
B7
XAPC
Anti-AChR Antibodies
RegulatoryT
-
AChR
Inflammatory environment
Why a chronic inflammation?
Are regulatory cells present and efficient?
Corticoids-
Percentage of CD4+CD25+ thymocytes Percentage of CD4+CD25+ thymocytes is unchanged in MG patientsis unchanged in MG patients
% CD25+ among CD4+ cells
0
10
20
30
MG patients
adultsnewborns
Controls
N.S.
N.S.
% CD25hi among CD4+ cells
0
2
4
6 N.S.
N.S.
MG patients
adultsnewborns
Controls
What about the function?
Ratio of CD4+CD25+/ CD25-
cells
MG
AdultNewborn
0
20
40
60
80
100
120
0 0.2 0.4 0.6 0.8 1 1.2% o
f p
rolif
era
tive r
esp
on
se
The suppressive function of CD4+CD25+ cells The suppressive function of CD4+CD25+ cells in MG patient thymus is severely impairedin MG patient thymus is severely impaired
(Balandina et al, Blood, 2005, 1% top citation)
Are peripheral CD4+CD25+ cells also defective?
Treg cells are also deficient in Treg cells are also deficient in PBMC, but at a lesser extentPBMC, but at a lesser extent
% P
rolif
erati
on
0
60
80
20
40
100
Controls MG%
Pro
lifer
ation
0
60
80
20
40
100
Controls MG
THYMUS PBMC
Is the phenotype of thymic and peripheral CD4+CD25+ cells different?
MG Control0
20
40
60
80
100%
CD
127ne
g/lo
w
MG Control0
10
20
30
40
50
60
% C
D127ne
g/lo
w
Expression of CD127neg/low in CD4+CD25+ cellsExpression of CD127neg/low in CD4+CD25+ cells
THYMUS PBMC
In the thymus or in PBMC of MG patients, there is no difference in the expression of IL-7R in the CD4+CD8+ population. What about FAS?
Compared phenotype of Treg cells Compared phenotype of Treg cells in the thymus and PBMC in the thymus and PBMC
***
**
*
In MG, CD95 (Fas) is highly increased in thymic, but not peripheral lymphocytes
CD95 in CD4+ thymocytes CD95 in CD4+ PBL
Compared phenotype of Treg cells Compared phenotype of Treg cells in the thymus and PBMC in the thymus and PBMC
in CD4+ Treg PBL
CONT MG CONT MG CONT MG CONT MG 0
25
50
75
100
CxCR3 CCR4 CxCR5 CCR7
ns
ns
ns
ns
% in
CD
4+
in CD4+ Treg THYMUS
CONT MG CONT MG CONT MG CONT MG 0
25
50
75
100
CxCR3 CCR4 CxCR5 CCR7
*
ns
***
ns
% in
CD
4+
In MG, both CXCR3 and CXCR5 are increased in thymic, but not peripheral CD4Treg cells
These results suggest that the thymic These results suggest that the thymic environment influences locally the thymic environment influences locally the thymic
CD4 and Treg cellsCD4 and Treg cells
Role of Thymic epithelial cells?Role of Thymic epithelial cells?
Major role in the differentiation of lymphocytes Overproduce pro-inflammatory cytokines in
MG
D6
Control thymic epithelial cells (TEC) maintain Treg Control thymic epithelial cells (TEC) maintain Treg phenotype of control CD4+CD25+ thymocytes phenotype of control CD4+CD25+ thymocytes
CD4 cells alone
CET+CD4
D6
TEC protect the Treg phenotypeTEC protect the Treg phenotypeC
D25 D
3/D
0 x
100
- TEC +
TEC
**
- TEC
+ TEC
FoxP
3 D
3/D
0 x
100
**
CD25 FoxP3
Total CD4+ cells
The effet is TEC specificThe effet is TEC specific
FoxP3
CD25
FoxP3
CD25
FoxP3
CD25
+
CET +
CACO2+
MITC
Cell
nu
mb
er
(Sta
nd
ard
ized
%)
Cell
nu
mb
er
(Sta
nd
ard
ized
%)
Cell
nu
mb
er
(Sta
nd
ard
ized
%)
Cell
nu
mb
er
(Sta
nd
ard
ized
%)
CD25
FoxP3
+
CET +
CACO2+
MITC
% C
on
trol
% C
on
trol
+ TEC+ MITC
FGM
42
80
+ TEC+ MITC
FGM
28
40
+ TEC+ CACO2
FGM
31
67
+ TEC+ CACO2
FGM
31
67
In contact with MG TEC, normal CD4+ In contact with MG TEC, normal CD4+ cells has a low suppressive abilitycells has a low suppressive ability
Teff
Treg
Teff
Treg
Co-culture(3 days)
Control CD4+
Cont TEC
MG TEC
% proliferation
Suppression assay
*
0
20
40
60
80
100
MG1 MG2
% o
f p
rolif
era
tio
n
In contact with normal TEC, MG CD4+ cells In contact with normal TEC, MG CD4+ cells have an increased suppressive ability have an increased suppressive ability
compared to cells before culturecompared to cells before culture
ConclusionsConclusions MG Treg are defective both in the thymus and at the
periphery (at a lesser extent). This is not due to higher number of Teff cells
TEC involved in Treg phenotype and functionMG TEC induce defective Treg
Normal TEC reverse partially the defect of MG Treg
Defects of Treg from MG patients probably linked to the inflammatory thymic environment, potentially reversible:
Cell therapy
Anti-acetylcholine receptor autoimmune response Anti-acetylcholine receptor autoimmune response occurs in the thymus of MG patientsoccurs in the thymus of MG patients
Cytokines
Th2 B
IL-4
T cellsV5.1Fas
resting T
activated T
MHC
B7
XAPC
Anti-AChR Antibodies
RegulatoryT
-
AChR
Inflammatory environment Why a chronic
inflammation? Are regulatory cells
present and efficient?
Collaborators and financial supportCollaborators and financial support
D. Nazzal F. Truffault J. Bismuth N. Kerlero de Rosbo
Thank you for your attentionThank you for your attention
0
100
200
300
400
500
Day0CD3+CD28
Day3 Day3 Day0CD3+CD28
Day3 Day3
Normal Thymus MG Thymus
Fox
P3 M
FI
M1M1
Normal cells MG cells
CD4+CD25neg cells from MG patients present a CD4+CD25neg cells from MG patients present a defect of FoxP3 regulationdefect of FoxP3 regulation
FoxP3 on human thymus sectionsFoxP3 on human thymus sections
Newborn thymus
Adult thymus
MG thymus
Keratin
FoxP3
CD21
FoxP3
Number of CD4+CD25+ is normal Phenotype of CD4+CD25+ cells
IL-7R normal mRNA FoxP3 decrease Fas increase
Function of CD4+ CD25+ cells Suppressive function defective
FoxP3
Cell number not decreased
Located in the medulla and GC
Regulation defective
CD4+25+ cells in the thymus of MG patients present CD4+25+ cells in the thymus of MG patients present phenotypic and functional abnormalitiesphenotypic and functional abnormalities
What are the consequences of Treg defects?
Overproduction of Il-1 and IL-6
• CD4+CD25+ Treg cells play a major role in prevention of autoimmunity, namely through the transcription factor, FoxP3, whose expression is highly correlated with the suppressive function.
• Numbers of thymic CD4+CD25+ Treg cells do not differ in MG patients and control individuals, while these cells present a severe functional defect in MG patients. A similar analysis in peripheral blood showed a similar defect in Treg function, although at a lower extent. In parallel, we investigated the phenotype of MG and control Treg. In the periphery, Treg cells from MG patients are very similar to control cells, while in the thymus Treg cells form MG patients express higher level of DR, Fas and CXCR3 compared to controls. These thymic-specific features suggest that thymic Treg cells may be influenced by unique interactions within the thymus. This leads us to analyze in more detail, the functional cross-talks between Treg and thymic epithelial cells (TEC) that participate to the development of self-tolerant T cells. We showed that TEC (from normal or MG thymus) are able to support the expression of CD25 and FoxP3 on CD4+ cells. Interestingly, the suppression activity of CD4+CD25+ issued from co-culture with TEC from MG patients, and not with non-MG TEC, was impaired as attested by functional assays.
• Altogether, these results suggest that functional Treg defects in MG patients are at least partially issued from defective interactions between developing T cells and TEC. Since we previously showed that TEC from MG patients overproduce IL-1 and IL-6, they could shift Treg cells towards a pro- inflammatory phenotype.
Control thymic epithelial cells (TECs) maintain Treg Control thymic epithelial cells (TECs) maintain Treg phenotype of control CD4+CD25+ thymocytes phenotype of control CD4+CD25+ thymocytes
CD25FoxP3
No.
of c
ells
(Sta
ndar
dize
d %
)
Treg
No.
of c
ells
(Sta
ndar
dize
d %
)Treg+ TEC
Treg Treg+ TEC
Regulatory CD4+CD25+ T cellsRegulatory CD4+CD25+ T cells
• The CD4+CD25+ Treg provide protection from T cell-mediated autoimmune disorders
• CD25+ cells are naturally anergic in vitro and they inhibit the proliferation of co-cultured CD4+CD25-
Organigramme Organigramme Chercheurs• Sonia Berrih-Aknin, DR INSERM• Rozen Le Panse-Ruskoné, CR CNRS• Nicole Kerlero de Rosbo, CDD EU• Nadine DRAGIN-MAMAVI, CDD EU• Angeline Gradolatto, CDD EU• Dani Nazzal, CDD EU• Sylvain Bougoin, CDD EU
ITA• Frederique Truffault, CCML• Jacky Bismuth, CCML• Perrine Cuffi, CDD ANR
• Margot Schmolinsky, CDD EU
Etudiants: Julia Weiss (PhD, ANR)
Angeline
Margot
Treg
IFN, virus et chemokine
Defective suppression of CD4+CD25+ cells Defective suppression of CD4+CD25+ cells 2 hypothesis
1) FoxP3 = transcription factor required for the suppressive function
2) Imbalance between Treg and activated effector cells
IL-7R
FAS
IL-7 R = CD127IL-7 R = CD127
J. Exp. Med, 2006
J. Exp. Med, 2006
Fas++ thymocytes accumulate in MG patients Fas++ thymocytes accumulate in MG patients with anti-AChR antibodieswith anti-AChR antibodies(Moulian et al., Blood, 1997)
1,3%
MG1, 15 yrs, 0 nM1,3%
Control, 17 yrs 1,2%
Fas
MG2, 36 yrs, 3 nM 4,1
%
14,7%
MG3, 19 yrs, 37 nM
Controls <1 1-10 >10 nM0
5
10
15
MG patients
N.S.
% o
f Fa
s++
thym
ocy
tes p<0.006 p<0.001
Fas expression in CD4+CD25+ cellsFas expression in CD4+CD25+ cells
% Fashi% Faslo
MGadultsnewborns
Controls
p<0.0001 p<0.0001
0
20
60
40
10
30
50
70p<0.0001N.S.
20
60
40
0
80
MGadultsnewborns
Controls
Fas lo = Treg?Fas hi = effector?
0
50
100
% o
f p
roliferativ
e r
esponse
CD25+CD25- + + +
+ FashiFaslo+ + ++ Fashi Faslo
Controls MG patients
Both CD4+CD25+ Faslo and Fashi are functionally suppressive in Both CD4+CD25+ Faslo and Fashi are functionally suppressive in controls but defectivecontrols but defective in MG patients in MG patients
Fas phenotype of CD4+CD25+ cellsFas phenotype of CD4+CD25+ cells(Balandina et al, Blood, 2005)
Thymocytes CD4+CD25+ thymocytes from MG patients
Increase of Fashi
Decrease of Faslo
CD4+CD25-
CD4+CD25+
FAS
59 %MFI=263
7 %FashiMFI=51
MFI=520 %
MFI=117
MFI=11
FashiMFI=9
MG
Adult
Newb