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Regulatory Lymphocytes of the Immune System.
II
Dr. C. PiccirilloCanada Research Chair
Department of Microbiology & ImmunologyMcGill University
MIMM-414ALecture 3- Oct. 25, 2006
CD25GITRCTLA-4Foxp3
ActivatedEffector
T cell
APC
__
TCR
+ Peripheraldifferentiation
signals
Thymic CD4Thymic CD4++ T cell pool T cell pool
Thymically-derived naturally-occurring
CD4+CD25+ Treg cells (nTreg )
Peripherally-induced CD4+ Treg cells
( iTreg )
Autoimmunity Transplantation Tumor Immunity
Infectious disease
TCR
Foxp3+
GITR+
CTLA-4+
CD25+
Piccirillo et al. Trends in Immunol. 2004.
NATURALLY-OCCURRING versus INDUCEDTreg cells in the immune system.
IL-10,TGF-1iDCVitDDexamethasone
CD4CD4++CD25CD25++ nTreg cells nTreg cellsMasterswitch of peripheral toleranceMasterswitch of peripheral tolerance
Self antigens Non-self antigensPathogens
TumorsAllergens
Grafts
FoxP3 transcription factor FoxP3 spontaneous mutations induces autoimmunity:
-IPEX in humans: Immunodysregulation, polyendocrinopathy, enteropathy,X-linked syndrome
-Scurfy in mice.
FoxP3-/- develop spontaneous autoimmunity- defective Treg cells
FoxP3 is preferentially expressed in CD4+CD25+ T cells
FoxP3 Tg have cellular frequency of CD4+CD25+ Treg cells.
FoxP3 Tg mice x CTLA-4-/- = resolved/delayed autoimmunity
FoxP3 retroviral transduction in non-regulatory CD4+CD25- T cells induces regulatory potential.
- Phenotypically and functionally similar to naturally occuring lineage.
Genes induced by FoxP3 remain unknown.
Fontenot et al.
More selective and faithful marker than CD25
nTregTeff cell
Antigen Presenting Cell
CD4+CD25+
CD4+ Teff
APC
CD4+
CD25+
• Requires TCR engagement
• Antigen non-specific
• Cell-cell contact dependent• Co-stimulation/APC independent• T-T suppressor synapse
• Suppress IL-2 mRNA in T cells.
• Suppression of effector functions
• proliferation• inflammatory cytokines• differentiation
• Effector molecules are unknown.
•Suppressive cytokines?
Cellular and molecular requirements of CD4+CD25+ nTreg cell suppressor function.
Suppressor Synapse
Mechanism of CD4+CD25+ regulatory T cell function ?
Contribution of Transforming Growth Factor 1 (TGF-1) ?
J.Exp. Med. 196:237-250.
EffectorT cell
CD4+
CD25+
Cytokines ?
Role of cytokines in CD4+CD25+ Treg cell-mediated suppression?
• IL-4, IL-10• Immunosuppressive effects on APC and T cells
•Suppression is cytokine independent in vitro• Cytokine neutralization
• Absence of cytokines in suppressor supernatants
• Cytokine-deficient Treg cells
• Transwell chamber experiments
CD4+CD45Rbhigh
CD4+CD45Rbhigh
CD4+CD45Rblow
ColitisNo colitis
(CD25+ subset)
SCID
CD4+CD25+ Treg cells control bacterial-driven intestinal inflammation.
Bacterially-driven, Th1 cell-mediated
Inflammatory bowel disease(IBD)Colitis
T cell infiltration of colon ->weight loss
Suppressor T cell-derived IL-10 needed.
Suppressor T cell-derived TGF-1?
Initial studies showed that anti-IL-10 or anti-TGF-1abrogated Treg-mediated suppression of disease.
Nakamura et al JEM 2001Membrane-bound TGF ?
Powrie et al. JEM 1994Simon Read et al. JEM 2000.
Requirement for TGF-TGF-1 1 ?
Potent immunosuppressive cytokine on various immune cell subsetsSuppression of T, B and DC responses: proliferation, cytokine, MHC/Ag
presentation and co-stimulation.Role is best exemplified in TGF-1 knock-out mice which die of a fulminant,
multi-organ, lymphoproliferative disease.
Piccirillo et al. J.Exp. Med. 196:237-250.
nTreg
TGF-TGF-11
RII
Smad3
TGF-R
DNRIITg
Smad3-/-
X
XTGF-TGF-11-/--/-
Y
Y
CD4+CD25- and CD4+CD25+ T cells produce TGF-1?
CD4+CD25-
CD4+CD25-
WT CD4+CD25+
CD4+CD25-
TGF-1-/-CD4+CD25+
No colitis
?
B6RAG-/-
3-7 day oldneonates
WT B6/Sv129 WT B6/Sv129TGF-1-/-
CD4+CD25+ Treg cell-mediated control of mucosal inflammation.
Mouse model of Inflammatory bowel disease (IBD)
Colitis• T cell infiltration of colon• Th1 response to gut bacteria• Weight loss
Colitis
Kullberg M., and C.A. Piccirillo Euro. J. Immunol. 2005
Is nTreg cell functionTGF- dependent in vivo?
CD25– CD25+
cells cells
— — WT —
WT WT
WT TGF-1-/-
— TGF-1-/-
80
90
100
110
0 10 20 30 40 50 60 70
Days post cells
Bo
dy
We
igh
t (%
of
da
y 4
we
igh
t)B
od
y w
eig
ht
(% o
f d
ay 4
wei
gh
t)
Days post cells
TGF-1-/- CD4+CD25+ nTreg cells suppress IBD.
TGF-1-/- CD4+CD25+ nTreg cells suppresscolonic inflammation.
AG
rad
e o
f in
flam
mat
ion
B
IFN
- /
G3P
DH
mR
NA
rat
io
CD25– cells — WT WT WT —
CD25+ cells — — WT TGF-1-/- TGF-1-/-
CD25– cells — WT WT WT —
CD25+ cells — — WT TGF-1-/- TGF-1-/-
C.
A.
D.
E.
B.A. B. C. D. E.
CD4+CD25-
WT CD4+CD25-
CD4+CD25+
Smad 3-/- CD4+CD25-
CD4+CD25+
Colitis
No colitis
?
B6RAG-/-
4-6 weeks old
WT B6/Sv129WT B6/Sv129
Smad3 -/-
FACS sort
CD4+CD25+-mediated regulation of Smad3-deficient effector T cells in vivo.
0
2
4
6
8
10
Gra
de
of
infl
am
ma
tio
n
CD25- cells – WT WT WT Smad3-/- Smad3-/- Smad3-/-
CD25+ cells – – WT Smad3-/- – WT Smad3-/-
A
B
Bo
dy
we
igh
t (%
of
da
y 4
we
igh
t)G
rad
e o
f in
fla
mm
ati
on
CD25– CD25+
— — WT Smad3-/- Smad3-/- WT Smad3-/- Smad3-/- WT WT Smad3-/- — WT —
Days post cells
Smad3-/- effector T cells are highly susceptible to suppression mediated by CD4+CD25+ T cells in vivo.
Powrie group observesabrogation of protection withTGFR-/- Effector T cells
Why?
CD45RBLow
CD4+CD25-
CD45RBHigh
+ TGF-1
- TGF-1
1:4 1:2 1:10
20
40
60
80% S
up
pre
ssio
n
Suppressor: Effector Cell
TGF-TGF-11
iTreg nTreg
Regulation of immune responses via Foxp3
induction
CD4+Foxp3+
IL-10IL-10++CD4+Foxp3Foxp3++
CD25-Rblow
Any role for TGF-1 in Treg responses?
Tissue-specific CD4+CD25+ mediated disease protection in the absence of IL-10.
CD4+CD25-
CD4+CD25-
CD4+CD25+ CD4+CD25-
IL-10-/- CD4+CD25+
GastritisIBD
No Gastritis
NoIBD
Nude
No GastritisIBD develops !IBD develops !
• Context-dependent regulation in vivo.– Tissue-specific differentiation of Treg?– Any role for bacteria?
• IBD is a bacterially-driven disease, not gastritis.
• Lessons from germ-free mice.
• Genetic background
• Subsets of CD4+CD25+ Treg?– Cytokine versus Contact– Adaptable to inflammatory milieu.– Induction of other Treg cells.
Control of immune responses by CD4+CD25+ regulatory T cells.
CD4+
CD8+
T cells
CD4+CD25+
RegulatoryT cells
Infectious disease
Immunity to intracellular pathogens ??
Belkaid/Piccirillo et al. Nature 420:502-7, 2002
100
103
104
105
106
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
0 5 10 15 20P
aras
ite
nu
mb
er
Lesio
n s
ize (mm
)
Weeks post-infection
Chronic phase : Transmits back to vector Resistant to re-infection. Life-long immunity : concomitant immunity
Site of immune pressure : IL-10IFN-IL-10-/- or anti-IL-10R -> Sterile cure.
Sile
nt
Acute
Chronic
Role for nTreg cells?
Weeks post-infection4 80
Th2 Non-healing
Les
ion
siz
e /
par
asit
e #
BALB/c
Susceptibility and resistance to Leishmania major infection
Healing Th1
B6
CD4+CD25+ CD4+CD25-
Chronic Dermal site
Cell Sorting
CD4+ nTreg cells accumulate in sites of chronic infection.
L.major infected DC
CytokineProduction
0
200
400
600
800
1000
1200
I-10
0
5000
1 104
1.5 10 4
IFN-
pg/
ml
CD4+CD25-
++++
CD4+CD25+
+
+
+
+
DCs
Infected DCs
45-60%
CD
25
CD4
8 months
CTLA-4 +Foxp3+GITR+CD45Rblow
CD4+CD25+ T cells from chronic sites are regulatory.
0
10000
20000
30000
40000
50000
60000
CP
M
0 10000200003000040000500000
50000
100000
150000
200000
CP
M
-IL-2 +IL-2
CD4+CD25+CD4+CD25-
CD4+CD25+
CD4+CD25-
0
3
6
9
12
15
IFN
- (
ng
/ml)
CD25- CD25-/CD25+CD25-/CD25+
Anti-IL-10R# CD4+CD25±
- -/+ -/+
IL10R
L.major Infected
macrophages
CD4+CD25+
CD4+CD25-
L. majorIntradermally
C57BL/6RAG-/-
1/10
Chronic site
CD4+CD25+T cells control immunity to pathogens. L
y5.
1
CD25
50%12 % 65 %
9 WksChronic
5 WksAcute
3 WksSilent
0
2
4
6
8
10
12
0 2 4 6 8 10 12
CD25-/CD25+
CD25-
RAG -/-
1. Rapid nTreg accumulation2. Prevent effector T cell functions.3. Promote susceptibility to infection4. Remain in chronic site5. Favor persistence of pathogen
IL-10+ Treg / IFN+ Teff cells
Model of cutaneous L.major infection.
Pa
ras
ite
nu
mb
er
/ea
rCD4+CD25+T cells from chronic sites prevent
anti-parasite effector T cell function.
CD4
IFN-
1
10
100
1000
104
105
106
107
RAG-/- CD25+ CD25- CD25-/CD25+
10/1
CD25-/CD25+
CD25-
CD25+
Lesi
on s
ize
Weeks post infection
IL-10 dependent and independent modes of disease control by nTreg cells.
-/+ WT
-/+ IL-10-/-0
0.5
1
1.5
2
0 2 4 6 8 10 12 14 16
CD25-
CD25-
RAG-/-
109
103
108
107
106
105
104
102
10
Par
asite
num
ber
CD25-CD25+
WT
CD25-CD25+IL-10-/-
3 103 103 10
Parasite persistence is required for immunity to re-infection
Implications :
•Parasite • Long-term maintenance of
infectious reservoirs.
•Host • Role of parasite
persistence in immunity ?
Homing of Treg cells to the infected sites
Preferential tropism for Treg cells to infected sites?
Chemokine-mediated selective recruitment of CD4+CD25+T cells ?
Journal of Experimental Medicine Oct. 2006
CCR5 gene expression
A.
B.
CCR5
Resting Activated
CCR5 is required for CD4+CD25+ nTreg cell chemo-attraction but not suppressive activity in vitro.
Gated on Foxp3+ cells
C.
0
40000
80000
120000
160000
CP
M
0 10000 20000 30000 40000 50000
# CD4+CD25+ T cells
CCR5-/-
WTD.
0
10
20
30
40
50
% M
igra
ting
CD
4+ T
ce
lls
CD4+Foxp3- CD4+Foxp3+
RANTES
MIP-1
MIP-1
CCR5-/- mice are resistant to L.major infection.
CCR5-/- CD4+CD25+ nTreg cells fail to promote parasite persistence.
Weeks post infection
0
0.5
1
1.5
0 1 2 3 4 5 6 7 8
WT
CD4+CD25+
102
103
104
105
+ +
Par
asit
e n
um
ber
7 weeks
Les
ion
siz
e (m
m)
WT CD4+CD25+
CCR5-/- CD4+CD25+
WT CD4+CD25-
+
-
-
+
CCR5-/-
CD4+CD25+101
+
--
0
10
20
30
40
50
60
70 WT
CCR5-/-
WT
CCR5-/-
WT
CCR5-/-
1.5 3 5.5 10
ND
% C
D4+
CD
25+
T c
ells
Weeks post-infection
ND
Spleen
Lymphnode
Skin38% 4%
CD
4+C
D2
5+ L
y5
.1+
3 weeks3 weeks
CD
4+C
D2
5+ L
y5
.2+
CD4+ effector T cells
WT CCR5-/-
CCR5 dependent homing of CD4+CD25+ Treg cells in sites of infection.
Summary• nTreg cells home preferentially to sites of
inflammation:
– nTreg cells express CCR5 and responds to its ligands.
– CCR5-mediated signals may drive the early recruitment of nTreg cells in sites of infection.
– CCR5 mediated homing into sites of pathogen infection regulates pathogen persistence.
– Pathogen persistence may itself provide a major benefit to the
host by maintaining life long immunity to re-infection.
– Blockade of CCR5 chemotaxis may hinder nTreg/Teff balance and provoke anti-pathogen immune responses.
– Mechanism of immune evasion ?
– Other receptors: CCR4 and CCR6 ( tumors and CNS homing)
CD4+ nTreg cell function in health and disease Diversification versus adaptability model
CD4+CD25+
nTreg
Self Autoimmunity
Foreign Pathogens
Tumors Grafts
Loss of tolerance
Increased immunity
Subset Diversification
Adaptability
Current Drug Targets 2006.
Genetic determinantsInnate signals
Adaptive signals
Teff
-