Les lymphocytes T régulateurs :
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Les lymphocytes T régulateurs :
du laboratoire à la clinique
I3: Immunology, Immunopathology, ImmunothérapyHopital Pitié Salpêtrière Paris
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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
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
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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.
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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)
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
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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
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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
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)
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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
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
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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 :
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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
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