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
CD4+CD25+ Treg cells
(nTreg )
Peripherally-induced
CD4+ Treg cells
( iTreg )
Foxp3+
GITR+
CTLA-4+
CD25+
TCR
TCR
CD25
GITR
CTLA-4
Foxp3
+ Peripheral
differentiation
signals
APC _ _
IL-10,
TGF-b1
iDC
VitD
Dexamethasone
Activated
Effector
T cell
Autoimmunity
Transplantation
Tumor Immunity
Infectious disease
Piccirillo et al. Trends in Immunol

3. CD4+CD25+ nTreg cells Masterswitch of peripheral tolerance
Immunity
Non-self antigens
Pathogens
Tumors
Allergens
Grafts
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 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.

5. More selective and faithful
Fontenot et al.
More selective and faithful
marker than CD25

6. Antigen Presenting Cell
Mechanism of CD4+CD25+ regulatory T cell function ?
Cellular and molecular requirements of CD4+CD25+ 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
CD4+CD25+
Suppressor Synapse
APC
CD4+
CD25+
CD4+
Teff

7. CD4+CD25+ Treg cell-mediated suppression?
Role of cytokines in
CD4+CD25+ Treg cell-mediated suppression?
CD4+
CD25+
Effector
T cell
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. 196:

8. CD4+CD25+ Treg cells control bacterial-driven intestinal inflammation.
CD4+CD45Rbhigh
CD4+CD45Rblow
(CD25+ subset)
CD4+CD45Rbhigh
Bacterially-driven,
Th1 cell-mediated
Inflammatory bowel disease
(IBD)
Colitis
T cell infiltration of colon ->weight loss
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?
Powrie et al. JEM 1994
Simon Read et al. JEM 2000.
Nakamura et al JEM 2001
Membrane-bound TGF ?

9. Potent immunosuppressive cytokine on various immune cell subsets
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.
RII Y
TGF-1
TGF-R
nTreg Y X
DNRIITg
Smad3-/-
TGF-1-/- X
Smad3
CD4+CD25- and CD4+CD25+
T cells produce TGF-b1?
Piccirillo et al. J.Exp. Med. 196:

10. TGF- dependent in vivo?
Is nTreg cell function
TGF- dependent in vivo?
CD4+CD25+ Treg cell-mediated control of mucosal inflammation. Mouse model of Inflammatory bowel disease (IBD)
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
CD4+CD25-
WT CD4+CD25+
CD4+CD25-
TGF-b1-/-CD4+CD25+
CD4+CD25- B6
RAG-/- ?
Colitis No
colitis
Kullberg M., and C.A. Piccirillo Euro. J. Immunol. 2005

11. TGF-1-/- CD4+CD25+ nTreg cells suppress IBD.
cells cells
— —
WT —
WT WT
WT TGF-b1-/-
— TGF-b1-/- 
Body weight (% of day 4 weight)   
Days post cells

12. TGF-1-/- CD4+CD25+ nTreg cells suppress colonic inflammation.B. 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-/-

13. CD4+CD25+-mediated regulation of
Smad3-deficient effector T cells in vivo.
WT B6/Sv129
Smad3 -/-
WT B6/Sv129
4-6 weeks old
FACS sort
WT CD4+CD25-
CD4+CD25+
Smad 3-/- CD4+CD25-
CD4+CD25+
CD4+CD25- B6
RAG-/-
Colitis ? No
colitis

14. Smad3-/- effector T cells are highly susceptible to
suppression mediated by CD4+CD25+ 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

15. Regulation of immune responses via Foxp3 induction
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
CD4+CD25-
CD4+Foxp3+
IL-10+
CD4+Foxp3+
CD25-Rblow
% Suppression
Suppressor: Effector Cell

16. Tissue-specific CD4+CD25+ 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 CD4+CD25+ Treg?
Cytokine versus Contact
Adaptable to inflammatory milieu.
Induction of other Treg cells.
CD4+CD25-
CD4+CD25+
CD4+CD25-
IL-10-/- CD4+CD25+
CD4+CD25-
Nude
Gastritis
IBD
No Gastritis
IBD develops ! No
Gastritis
IBD

17. Control of immune responses by CD4+CD25+ regulatory T cells.
Infectious disease
Immunity to
intracellular pathogens ? ?
CD4+CD25+
Regulatory
T cells
Belkaid/Piccirillo et al. Nature 420:502-7, 2002

18. Susceptibility and resistance to Leishmania major infection
Non-healing
Healing
Th2
Th1 10 6
0.2
0.4
0.6
0.8 1
1.2
1.4
1.6
1.8 B6
Lesion size / parasite #
Parasite number
Acute 10 5
Silent
Lesion size (mm) 10 4
Chronic
103
BALB/c 4 8
100 5 10 15 20
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 -> Sterile cure.
Role for nTreg cells?

19. CD4+ nTreg cells accumulate in sites of chronic infection.1 2
Chronic
Dermal site
I-10
CD4+CD25+ + 1
45-60% 8
CD25
8 months 6
CTLA-4 +
Foxp3+
GITR+
CD45Rblow 4 2
pg/ml
CD4+CD25- +
CD4 4 1 . 5 1
IFN-g
Cell
Sorting 4 1 1
CD4+CD25+
CD4+CD25- 5
Cytokine
Production
DCs
Infected DCs
L.major infected DC

20. CD4+CD25+ T cells from chronic sites are regulatory.
10000
20000
30000
40000
50000
60000
CPM
100000
150000
200000
-IL-2
+IL-2
CD4+CD25+
CD4+CD25-
L.major
Infected
macrophages
IL10R
# CD4+CD25±
/ /+
Chronic site
CD4+CD25+
L. major
Intradermally
C57BL/6
RAG-/-
1/10
CD4+CD25-

21. IL-10+ Treg / IFNg+ Teff cells
CD4+CD25+T cells control immunity to pathogens.
Model of cutaneous L.major infection.
3 Wks
Silent
5 Wks
Acute
9 Wks
Chronic
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
12 %
65 %
50%
Ly5.1
CD25

22. CD4+CD25+T cells from chronic sites prevent
anti-parasite effector T cell function.
CD25+
IFN-g
CD25+ 1 10
100
1000 4 5 6 7
CD25-/
CD25+
10/1
Parasite number /ear
CD25-
CD25-
CD25-/
CD25+
RAG-/-
CD4

23. IL-10 dependent and independent
modes of disease control by nTreg cells.
0.5 1
1.5 2
-/+ IL-10-/-
Lesion size
-/+ WT
Parasite persistence is required for
immunity to re-infection
Implications :
CD25- 2 4 6 8 10 12 14 16
Parasite
Long-term maintenance of
infectious reservoirs.
Host
Role of parasite
persistence in immunity ?
Weeks post infection
109
108
107
RAG-/-
106
CD25-
CD25+ WT
CD25-
CD25+
IL-10-/-
Parasite number
105
104      
103
102
CD25-    10        

24. 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

25. CCR5 CCR5 is required for CD4+CD25+ nTreg cell chemo-attraction
but not suppressive activity in vitro.
CCR5 gene expression A. C. D. B.
Resting
Activated
Gated on
Foxp3+ cells
CCR5

26. CCR5-/- mice are resistant to L.major infection.

27. CCR5-/- CD4+CD25+ nTreg cells fail to promote parasite persistence.
0.5 1
1.5
102
103
104
105
7 weeks WT
CD4+CD25+
Lesion size (mm)
Parasite number
CCR5-/-
CD4+CD25+
101 + +
WT CD4+CD25- + 1 2 3 4 5 6 7 8
Weeks post infection
WT CD4+CD25+ + - -
CCR5-/- CD4+CD25+ - + -

28. CCR5 dependent homing of CD4+CD25+ Treg cells in sites of infection.
3 weeks WT
CCR5-/- 10 20 30 40 50 60 70 WT
Skin
CCR5-/-
38% 4% WT
Lymph
node
CD4+CD25+ Ly5.1+
CD4+CD25+ Ly5.2+
CCR5-/- WT
Spleen
% CD4+CD25+ T cells
CCR5-/-
CD4+ effector T cells ND ND
1.5 3
5.5 10
Weeks post-infection

29. 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)

30. CD4+ nTreg cell function in health and disease Diversification versus adaptability model
Subset
Diversification
CD4+CD25+
nTreg
Adaptability -
Foreign
Pathogens
Tumors
Grafts
Self
Autoimmunity
Teff
Loss of
tolerance
Genetic determinants
Innate signals
Adaptive signals
Increased
immunity
Current Drug Targets 2006.