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