Regulatory Lymphocytes of the Immune System. II

1
Regulatory Lymphocytes of the Immune System.II
Dr. C. Piccirillo Canada Research
Chair Department of Microbiology
Immunology McGill University MIMM-414A Lecture
3- Oct. 25, 2006
2
NATURALLY-OCCURRING versus INDUCED Treg cells in
the immune system.
Thymic CD4 T cell pool
Thymically-derived naturally-occurring CD4CD25
Treg cells (nTreg )
Peripherally-induced CD4 Treg cells ( iTreg )
Foxp3
GITR
CTLA-4
CD25
TCR
TCR
Peripheral differentiation signals
CD25 GITR CTLA-4 Foxp3
APC
_
_
IL-10, TGF-b1 iDC VitD Dexamethasone
Activated Effector T cell
Autoimmunity Transplantation Tumor Immunity
Infectious disease
Piccirillo et al. Trends in Immunol. 2004.
3
CD4CD25 nTreg cellsMasterswitch of peripheral
tolerance
Immunity
Tolerance
Non-self antigens Pathogens Tumors Allergens Graft
s
Self antigens
4
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 CD4CD25 T
  cells
• FoxP3 Tg have ? cellular frequency of CD4CD25
  Treg cells.
• FoxP3 Tg mice x CTLA-4-/- resolved/delayed
  autoimmunity
• FoxP3 retroviral transduction in non-regulatory
  CD4CD25- T cells
• induces regulatory potential.
• - Phenotypically and functionally similar to
  naturally occuring lineage.
• Genes induced by FoxP3 remain unknown.

5
Fontenot et al.
More selective and faithful marker than CD25
6
Mechanism of CD4CD25 regulatory T cell
function ?
Cellular and molecular requirements of CD4CD25
nTreg cell suppressor function.

• 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?

nTreg
Teff cell
Antigen Presenting Cell
CD4CD25
Suppressor Synapse
APC
CD4 Teff
CD4 CD25
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Role of cytokines in CD4CD25 Treg
cell-mediated suppression?
Effector T cell
CD4 CD25
Cytokines ?

• 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

Contribution of Transforming Growth Factor ?1
(TGF-?1) ?
J.Exp. Med. 196237-250.
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CD4CD25 Treg cells control bacterial-driven
intestinal inflammation.
CD4CD45Rbhigh CD4CD45Rblow
(CD25 subset)
Bacterially-driven, Th1 cell-mediated Inflammator
y bowel disease (IBD) Colitis T cell infiltration
of colon -gtweight loss
CD4CD45Rbhigh
SCID
Colitis
No colitis
Initial studies showed that anti-IL-10 or
anti-TGF-b1 abrogated Treg-mediated suppression
of disease.
Suppressor T cell-derived IL-10 needed.
Suppressor T cell-derived TGF-?1?
Nakamura et al JEM 2001 Membrane-bound TGF ?
Powrie et al. JEM 1994 Simon Read et al. JEM 2000.
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Requirement for TGF-?1 ? Potent
immunosuppressive cytokine on various immune cell
subsets Suppression of T, B and DC responses
proliferation, cytokine, MHC/Ag presentation and
co-stimulation. Role is best exemplified in
TGF-b1 knock-out mice which die of a fulminant,
multi-organ, lymphoproliferative disease.
Y
TGF-?R
TGF-?1
nTreg
Y
X
DNRIITg Smad3-/-
TGF-?1-/-
X
Smad3
CD4CD25- and CD4CD25 T cells produce TGF-b1?
Piccirillo et al. J.Exp. Med. 196237-250.
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CD4CD25 Treg cell-mediated control of mucosal
inflammation. Mouse model of Inflammatory bowel
disease (IBD)
Is nTreg cell function TGF-? dependent in
vivo?
WT B6/Sv129
WT B6/Sv129 TGF-b1-/-
3-7 day old neonates

• Colitis
• T cell infiltration of colon
• Th1 response to gut bacteria
• Weight loss

CD4CD25- WT CD4CD25
CD4CD25- TGF-b1-/-CD4CD25
CD4CD25-
B6 RAG-/-
?
Colitis
No colitis
Kullberg M., and C.A. Piccirillo Euro. J.
Immunol. 2005
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TGF-?1-/- CD4CD25 nTreg cells suppress IBD.
CD25 CD25 cells cells
WT WT
WT WT TGF-b1-/-
TGF-b1-/-
?
Body weight ( of day 4 weight)
?
?
?
Days post cells
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TGF-?1-/- CD4CD25 nTreg cells suppresscolonic
inflammation.
B.
A.
A
A.
B.
C.
D.
E.
Grade of inflammation
C.
D.
CD25 cells WT WT
WT CD25 cells
WT TGF-b1-/- TGF-b1-/-
B
E.
IFN-g / G3PDH mRNA ratio
CD25 cells WT WT
WT CD25 cells
WT TGF-b1-/- TGF-b1-/-

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CD4CD25-mediated regulation of Smad3-deficient
effector T cells in vivo.
WT B6/Sv129 Smad3 -/-
WT B6/Sv129
4-6 weeks old
FACS sort
WT CD4CD25- CD4CD25
Smad 3-/- CD4CD25- CD4CD25
CD4CD25-
B6 RAG-/-
Colitis
?
No colitis
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Smad3-/- effector T cells are highly susceptible
to suppression mediated by CD4CD25 T cells in
vivo.
A
CD25 CD25
WT Smad3-/- Smad3-/- WT
Smad3-/- Smad3-/- WT WT
Smad3-/- WT
?
Body weight ( of day 4 weight)
?
?
?
?
?
B
Days post cells
Powrie group observes abrogation of protection
with TGFR-/- Effector T cells Why?
Grade of inflammation
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Any role for TGF-b1 in Treg responses?
TGF-b1 iTreg nTreg Regulation of
immune responses via Foxp3 induction
TGF-b1
- TGF-b1
CD45RBLow
CD45RBHigh
CD4CD25-
CD4Foxp3 IL-10
CD4Foxp3
CD25-Rblow
Suppression
Suppressor Effector Cell
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Tissue-specific CD4CD25 mediated disease
protection in the absence of IL-10.

• 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 CD4CD25 Treg?
• Cytokine versus Contact
• Adaptable to inflammatory milieu.
• Induction of other Treg cells.

CD4CD25- CD4CD25
CD4CD25- IL-10-/- CD4CD25
CD4CD25-
Nude
Gastritis IBD
No Gastritis IBD develops !
No Gastritis No IBD
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Control of immune responses by CD4CD25
regulatory T cells.
CD4 CD8 T cells
Infectious disease Immunity to intracellular
pathogens ?
?
CD4CD25 Regulatory T cells
Belkaid/Piccirillo et al. Nature 420502-7, 2002
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Susceptibility and resistance to Leishmania
major infection
Non-healing
Healing
Th2
Th1
6
10
B6
Lesion size / parasite
Parasite number
Acute
Silent
Lesion size (mm)
Chronic
103
BALB/c
100
0
4
8
Weeks post-infection
Weeks post-infection

• Chronic phase
• Transmits back to vector
• Resistant to re-infection.
• Life-long immunity concomitant immunity
• Site of immune pressure IL-10?IFN-?
• IL-10-/- or anti-IL-10R -gt Sterile cure.

Role for nTreg cells?
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CD4 nTreg cells accumulate in sites of chronic
infection.
1
2
0
0
I?-10
Chronic Dermal site
1
0
0
0
45-60
8
0
0
CD25
8 months
6
0
0
4
0
0
CTLA-4 Foxp3 GITR CD45Rblow
2
0
0
pg/ml
0
CD4
4
1
.
5

1
0
IFN-g
Cell Sorting
4
1

1
0
CD4CD25
CD4CD25-
5
0
0
0
Cytokine Production
0
DCs
Infected DCs
L.major infected DC
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CD4CD25 T cells from chronic sites are
regulatory.
L.major Infected macrophages
?IL10R
CD4CD25
- -/ -/
Chronic site
L. major Intradermally C57BL/6 RAG-/-
CD4CD25
1/10
CD4CD25-
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CD4CD25T cells control immunity to pathogens.
Model of cutaneous L.major infection.
9 Wks Chronic
5 Wks Acute
3 Wks Silent

• 1. Rapid nTreg accumulation
• 2. Prevent effector T cell functions.
• 3. Promote susceptibility to infection
• 4. Remain in chronic site
• 5. Favor persistence of pathogen
• IL-10 Treg / IFNg Teff cells

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65
50
Ly5.1
CD25
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CD4CD25T cells from chronic sites prevent
anti-parasite effector T cell function.
IFN-g
CD25
Parasite number /ear
CD25-
CD25-/ CD25
RAG-/-
CD4
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IL-10 dependent and independent modes of disease
control by nTreg cells.
Lesion size
-/ WT
Parasite persistence is required for immunity
to re-infection
Implications
CD25-

• Parasite
• Long-term maintenance of
• infectious reservoirs.
• Host
• Role of parasite
• persistence in immunity ?

Weeks post infection
109
3 10
3 10
3 10
108
107
RAG-/-
106
CD25- CD25 WT
CD25- CD25 IL-10-/-
105
Parasite number
?
104
?
?
?
?
?
103
CD25-
102
?
?
?
10
?
?
?
?
?
?
?
?
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Homing of Treg cells to the infected sites
Preferential tropism for Treg cells to infected
sites? Chemokine-mediated selective
recruitment of CD4CD25T cells ?
Journal of Experimental Medicine Oct. 2006
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CCR5 is required for CD4CD25 nTreg cell
chemo-attraction but not suppressive activity in
vitro.
CCR5 gene expression
A.
C.
D.
B.
Resting
Activated
Gated on Foxp3 cells
CCR5
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CCR5-/- mice are resistant to L.major infection.
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CCR5-/- CD4CD25 nTreg cells fail to promote
parasite persistence.
7 weeks
WT CD4CD25
Lesion size (mm)
Parasite number
CCR5-/- CD4CD25
101



WT CD4CD25-

-
Weeks post infection
-
WT CD4CD25
-

-
CCR5-/- CD4CD25
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CCR5 dependent homing of CD4CD25 Treg cells in
sites of infection.
3 weeks
WT
CCR5-/-
WT
Skin
38
4
CCR5-/-
Lymph node
WT
CD4CD25 Ly5.1
CD4CD25 Ly5.2
CCR5-/-
WT
Spleen
CD4CD25 T cells
CCR5-/-
CD4 effector T cells
ND
ND
3
10
1.5
5.5
Weeks post-infection
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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)

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CD4 nTreg cell function in health and disease
Diversification versus adaptability model
Subset Diversification
CD4CD25 nTreg
Adaptability
-

• Foreign
• Pathogens
• Tumors
• Grafts

• Self
• Autoimmunity

Teff
Loss of tolerance
Genetic determinants Innate signals Adaptive
signals
Increased immunity
Current Drug Targets 2006.