Les lymphocytes T régulateurs : du laboratoireà la clinique
Transcript of Les lymphocytes T régulateurs : du laboratoireà la clinique
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Les lymphocytes T régulateurs :
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Les lymphocytes T régulateurs :
du laboratoire à la clinique
I3: Immunology, Immunopathology, ImmunothérapyHopital Pitié Salpêtrière Paris
© STI 2010
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Treg inhibit T cell proliferation in vitro
C57BL/6 responding T cells
BALB/C APCs
Hoffmann et al J.Exp.Med.2002
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Treg prevent AID
GastritisOophoritis
++++++
Impact
CD25
Sakaguchi. J.Immunol.1995
BALB/c nude reconstituted with CD4+CD25- T cells
OophoritisThyroiditis
SialoadenitisAdrenalitis
Insulitis
++++++++/-+/-
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CD25+
GastritisOophoritis
++++++
Impact
Treg prevent AID
Sakaguchi. J.Immunol.1995
OophoritisThyroiditis
SialoadenitisAdrenalitis
Insulitis
++++++++/-+/-BALB/c nude reconstituted with
CD4+CD25- T cells
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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
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Phenotypic characterization of Treg
• 5-10% of CD4+
• Constitutively express CD25 et CD62L
• and Foxp3
CD25 CD25
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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
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Treg in human ?Treg in human ?
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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
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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.
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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)
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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
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Treg and alloreactivity
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Do allo-Ag-specific Treg exist?
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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
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Alloreactivity following allo-HSCT
RD
Graft versus host disease (GVHD)
Anti-leukemic effect (GVL) RD
Host versus graft reaction (HVG)
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The T cell dilemma
Immune reconstitution
engraftmentDonor T cells
beneficial effects
anti-leukemic effect
Graft-versus-host disease
deleterious effects
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Modulating the immune response in vivo
���� use of CD 4+CD25+ immunoregulatory T cells���� use of CD 4+CD25+ immunoregulatory T cells
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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
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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
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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
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What do we know in humans ?What do we know in humans ?
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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
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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
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“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
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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
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Treg depletion to improve alloreactivity?
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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
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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
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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
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Procédure et contrôle qualitéde la déplétion des Treg
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Laboratoire de production
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Functional analysis
Allogeneic activation
3
Polyclonal activation
3
D 0D 6 (CULTURE)
0
1
2
FN
/FT
0
1
2
FN
/FT
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DLI-Treg : 11 patients enrolled
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Patients :
© STI 2010
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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
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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)
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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