Les lymphocytes T régulateurs : du laboratoireà la clinique

Les lymphocytes T régulateurs :

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Les lymphocytes T régulateurs :

du laboratoire à la clinique

jose.cohen@chups.jussieu.fr

I3: Immunology, Immunopathology, ImmunothérapyHopital Pitié Salpêtrière Paris

© STI 2010

Treg inhibit T cell proliferation in vitro

C57BL/6 responding T cells

BALB/C APCs

Hoffmann et al J.Exp.Med.2002

Treg prevent AID

GastritisOophoritis

++++++

Impact

CD25

Sakaguchi. J.Immunol.1995

BALB/c nude reconstituted with CD4+CD25- T cells

OophoritisThyroiditis

SialoadenitisAdrenalitis

Insulitis

++++++++/-+/-

CD25+

GastritisOophoritis

++++++

Impact

Treg prevent AID

Sakaguchi. J.Immunol.1995

OophoritisThyroiditis

SialoadenitisAdrenalitis

Insulitis

++++++++/-+/-BALB/c nude reconstituted with

CD4+CD25- T cells

CD25+

GastritisOophoritis

HighHigh

Incidence

Treg prevent AID

Sakaguchi. J.Immunol.1995

OophoritisThyroiditis

SialoadenitisAdrenalitis

InsulitisGN

HighModerateModerate

LowLowLow

BALB/c nude reconstituted with CD4+CD25- T cells

© STI 2010

Phenotypic characterization of Treg

• 5-10% of CD4+

• Constitutively express CD25 et CD62L

• and Foxp3

CD25 CD25

Treg suppress auto-immune diseases

Foxp3-DTR knock-in mice

Diphteria toxin

T cell activationT cell activationIncrease of T cells, B cells, APC within 7 days

Autoimmune syndrome within 20 days

Lahl et al. JEM 2007Kim et al. Nat Immunol 2007

Treg in human ?Treg in human ?

Comparison mice and human

101

102

103

104F

L3-H

: CD

4 P

erC

P

mice human

100 101 102 103 104

FL4-H: CD25 APC

100

101

102

103

104

FL2

-H: F

oxp3

PE

100 101 102 103 104

FL4-H: CD25 APC

100

CD25

CD25 Miyara et al. JEM 2006

Syndrome IPEX and Treg in human

Multi-organ autoimmune syndrom : endocrine tissus (diabetis, thyroïdite), gut, skin.

Treg deficiency due to a mutation in Foxp3.Treg deficiency due to a mutation in Foxp3.

© STI 2010

Treg deficit in PBC in AID

Quantitatif

IPEX

Juvenil Arthritis

Active forms of lupus

Vascularite associée à l’infection HCV

Qualitatif

Sclérose en plaque

Polyarthrite rhumatoïde

Poly-endocrinopathie auto-immune (APS-II)

Mode of action of Treg

LyT

LyT

LyT �Secretion of

inhibitory IL10

LyTCD39/CD73

A2A

cAMP

Déprivation

Adenosine

Cell contact

Treg

LyT

LyTLyT

LyT

�GrzB/A:Pfr

CMH IILAG-3

CD80/86

CTLA-4

IDO

mTGFβ

TGFβ

inhibitory cytokines

IL35CD25

Déprivationcytokine

Interaction with DCsAdapted from Vignali, Eur.J.Immunol, 2008

Treg and alloreactivity

Do allo-Ag-specific Treg exist?

Treg can induce an organ specific tolerancein organ transplantation

CD25+ purification Transfert

rejectionC

Type AB

« tolerized »

Type B specific inducton of tolerance

Type A

CD25+ purification Transfert

toleranceB

Hara et al. 2001. JI 166:3789Gregori et al. 2001. JI 167:1945

Type A

© STI 2010

Alloreactivity following allo-HSCT

RD

Graft versus host disease (GVHD)

Anti-leukemic effect (GVL) RD

Host versus graft reaction (HVG)

The T cell dilemma

Immune reconstitution

engraftmentDonor T cells

beneficial effects

anti-leukemic effect

Graft-versus-host disease

deleterious effects

Modulating the immune response in vivo

���� use of CD 4+CD25+ immunoregulatory T cells���� use of CD 4+CD25+ immunoregulatory T cells

depletion

Teff

Role of Treg in allo-HSCT

addition

Teff+

Treg-

Cohen et al J.Exp.Med.2002Taylor et al Blood.2002

Cohen et al J.Exp.Med.2002 Taylor et al Blood.2002

days post transplantation

0

20

40

60

80

100

0 10 20 30 40 50 60

% survival Treg depleted T cell

Total T cells

Taylor et al Blood.2002 Taylor et al Blood.2002Hoffmann et al J.Exp.Med.2002

days post transplantation

0 10 20 30 40 50 600

20

40

60

80

100Total T cells + TregTotal T cells

% survival

Quantitative and qualitative aspects

Treg represent 5% of CD4+ T lymphocytes

-> necessary to expand them in vitro for clinical a pplication

-> is it possible to select alloantigen specific Tr eg?

- to improve their effect

- to be more specific and, consequently to favor imm une

reconstitution from non alloreactive donor T cells

© STI 2010

80

100

% o

f sur

viva

l

GMO

sTreg

survival

rsTreg more efficient than polyTreg

0

20

40

60

0 20 40 60 80 100

% o

f sur

viva

l

Days after transplantation

polyTreg

What do we know in humans ?What do we know in humans ?

Number study

of patients identification of Treg conclusion

40 CD4+ CD25hi T cells (cytometry) More than 100 days post-graft, patients with cGVHD have Clark et al.

elevated numbers of CD4 + CD25hi T cells. ref. 22

54 CD4+ CD25hi T cells (cytometry) cGVHD does not correlates with the numbers Sanchez et al.

of CD4+ CD25hi T cells in grafted patients. ref. 23

34 Foxp3 (RT-qPCR) GVHD correlates with low Foxp3 expression level Miura et al.

in PBMCs of grafted patients. ref. 24

Clinical studies in allo-HSCT in grafted patients

57 CD4+ CD25+ T cells (cytometry) Patients with cGVHD have reduced frequencies of Zorn et al.

and Foxp3 (RT-qPCR) CD4+ CD25+ and Foxp3-expressing T cells. ref. 25

These cells are functionnaly suppressive in vitro.

31 CD4+CD25+ T cells (cytometry) and Foxp3 (RT-qPCR) The number of Foxp3 expressing CD4+CD25+ T cells Meignin et al.

does not correlate with GVHD in grafted patients ref. 27

49 Foxp3+ cells (immunostaining) Deficit of Foxp3 + cells in the intestine Rieger et al.

of patients with GVHD. ref. 28

32 CD4+Foxp3+ T cells (cytometry) High numbers of CD4 +Foxp3+ T cells Rezvani et al.

in the blood of grafted patients ref. 29

are associated with a reduced risk to develop GVHD.

Cohen & Boyer. Current opinion in Immunol. 2006

CD4+CD25hi CD4+FOXP3+

CD4+CD25high

7

8

CD4+FoxP3+

7

8

Treg in HSCT

no GVHD a-GVHD0

1

2

3

4

5

6

7

% o

f C

D4+

T c

ells

CD4+CD25high

no GVHD a-GVHD0

5

10

15

abso

lute

num

ber

of T

cel

lsu

btyp

es in

fuse

d (x

105 /k

g)

CD4+FoxP3+

no GVHD a-GVHD0

5

10

15

abso

lute

num

ber

of T

cel

lsu

btyp

es in

fuse

d (x

105 /k

g)no GVHD a-GVHD

0

1

2

3

4

5

6

7

% o

f C

D4+

T c

ells

Rosenzwajg et al.submited

“purification” of human Treg under GMP conditions

-primary depletion of CD19+ B cells

-3 selection cycles for CD25

2 clinical trials : Edinger groupMartelli group

50% of CD25high cells Foxp3+

with suppressive functions (50%suppression ratio 1/1)

© STI 2010

Outcome

ConditionningTreg infusion

Day 3CD34+ cells+ 2.106/kg T cell

Clinical scheme for Martelli group

ConditionningTreg infusion

2.106/kg

• 22 patients included• 3 GVHD• 6 patients died from infections• improved immune reconstitution

Treg depletion to improve alloreactivity?

Treg quantification in DLI

De Champs de Saint B.

0,1

1

%m-Bcr/GUS

Ph1+ ALL

DLI n°1106 CD3/kg

DLI n°2108 CD3/kg

Allo-HSCT

0,0001

0,001

0,01

sept-00 nov-00 janv-01 mars-01 mai-01 juil-01 sept-01 nov-01 janv-02 mars-02

• Far from the cytokine storm

• in non lymphopenic recipients

1

2

3

4

5

p=0.04 p=0.04

5

10

15

p=0.04

% of different donor CD4+

T cell subtypes

FOXP3+CD25high FOXP3+CD127low/neg CD4+ CD127low/neg

Treg in DLI

Donor regulatory T-cells identified by FoxP3 expression but also by the membranous CD4+CD127low/neg phenotype influence graft-versus-tumor effect after donor lymphocyte infusion.”

Hicheri et al., Journal of Immunotherapy : In press

no durable CR no durable CR

0

no durable CR

0

% of different donor CD

• p=0.04

•Trends of GVHD

Treg depletedDLI

2.106<dose<2.107

2.107<dose<108

CD3+/kgAllogeneic

hematopoietic stemLeukemia relapse

donor lymphocyte infusion

How to improve graft versus leukemia effect in donor lymphocyte infusion

hematopoietic stemcell transplantation

insufficient response& relapse

© STI 2010

Procédure et contrôle qualitéde la déplétion des Treg

Laboratoire de production

Functional analysis

Allogeneic activation

3

Polyclonal activation

3

D 0D 6 (CULTURE)

0

1

2

FN

/FT

0

1

2

FN

/FT

DLI-Treg : 11 patients enrolled

Patients :

© STI 2010

Conclusions

• we have demonstrated the feasibility of the Treg depletion from DLI

under clinical grade

• an increased alloreactivity in vitro observed after 6 days of culture

• absence of toxicity of the Treg depleted DLI in grafted patients• absence of toxicity of the Treg depleted DLI in grafted patients

• 3/11 patients developed clear manifestations of increased

alloreactivity

• 1 patient is in complete remission after 2,5 years

Perspectives

To develop alternative procedure to deplete Treg (CD127)

To combine Treg depletion with non myeloablative but

lymphodepleting chemotherapy (S. Rosenberg in K, Blazar in

DLI)DLI)

2 Clinical departments:

Henri Mondor Créteil (Sébastien Maury) & Pitié Salpêtrière, Paris (Nathalie Dhédin

et JP Vernant)

1 Biological department

Department of Biotherapy, Pitié-Salpêtrière,Paris

Financial support: Assistance Publique - Hôpitaux de Paris

INSERM - INCA - DHOS

I3: Immunology, Immunopathology, ImmunothérapyHopital Pitié Salpêtrière Paris

David klatzmann© STI 2010