1. Regulatory Lymphocytes of the Immune System.
Dr. C. Piccirillo
Canada Research Chair
Department of Microbiology & Immunology
McGill University
MIMM-414A
Lecture 2- Oct. 23, 2006

2. Induced regulatory T cells Peripherally-induced
Thymic CD4+ T cell pool
Peripherally-induced
CD4+ Treg cells
( iTreg )
TCR
+ Peripheral
differentiation
signals
APC _
Activated
Effector
T cell
Autoimmunity
Transplantation
Tumor Immunity
Infectious disease
Piccirillo et al. Trends in Immunol

3. A network of CD4+ regulatory T cells control immune reponses.
A. INDUCED - immunization, experimental manipulation…
NK-T cells
CD8+ T cells
 T cells
DN T cells
CD4+
Th1 cell-mediated cross-regulation of Th2 cells (IL-4 <-> IFN)
Th3 - Induced by oral tolerance
TGF-1 producing (IL-10) and TGF-1 dependent effect
Tr1 - Induced by Ag + IL-10 and IL-10 dependent effect

4. B. NATURALLY-OCCURRING Peripherally-induced
Endogenous in naïve repertoire: professional
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 _ _
Activated
Effector
T cell
Autoimmunity
Transplantation
Tumor Immunity
Infectious disease
Piccirillo et al. Trends in Immunol

5. Lymph nodes from a 2 month old BALB/c
Journal of Immunology :1151
Lymph nodes from a
2 month old BALB/c

6. Naturally-occurring CD4+ regulatory T cells.
Autoimmunity
Dominant self-tolerance
Unique lineage of CD25+ (IL-2R) T cells
1-10% of thymic or peripheral CD4+ T cells
In resting, endogenous T cell repertoire
Rodents, human and non-human primates
Allergy
Infectious
disease
CD4+CD25+
Treg cells
Tissue
regeneration
Transplantation
Mucosal
Immunity
Depletion
Day3 thymectomy
Genetic anomalies
Irradiation
Infections
Drugs
Gastritis
Oophritis
Orchitis
Thyroiditis
Pancreatitis
Colitis
Auto Abs
Normal
Immune
responses
Tumor
Immunity

7. CD4+CD25+ nTreg cells Masterswitch of peripheral tolerance
Immunity

8. Reliable biomarkers for CD4+CD25+ regulatory T cells?
GITR
CTLA-4
CD4+CD25+
Regulatory
T cell
NO marker after T cell activation.
Only in the naïve T cell repertoire.
Need better marker.
Galectin-1
CD103
Ly6
4-1BB
Neuropilin-1
Unidentified
X,Y,Z ??
OX-40
Piccirillo et al. Trends in Immunology July 2004

9. T cell activation induces expression of functional T cell surface molecules
Activated
CD4+ T cell
MHC/peptide
CD40L
TCR
TCR
CD25
Resting
CD4+ T cell
APC
CD69
CD44
CD25 is not a marker of Treg after T cell activation.
Only in the naïve T cell repertoire.

10. Phenotypic and functional characteristics
Treg cells are partially-activated cells
Treg cells are anergic.
Hyporesponsive to TCR stimulation : restored by IL-2.
Do not produce IL-2, IL-4, IFN-, IL-10 (), TGF1 ()

11. CD4+CD25+ regulatory T cells suppress T cell functions
CD4+CD25+ regulatory T cells suppress T cell functions. Consensus elements
24h
CD8+
Proliferation
Cytokine Production
CD8+/-
CD8+/+
CD4+ or CD8+
T cells
CPM
48h
# CD4+CD25- (  ) or CD4+CD25+ ( ) T cells
Requirement for TCR engagement
Antigen non-specific & MHC unrestricted
Induce cell cycle arrest (G1-S transition)
(ng/ml)
IFN- g
IFN-
CD8+
CD8+/-
CD8+/+
CD4+CD25+ Immunoregulatory T Cells Suppress Polyclonal T Cell Activation In Vitro by Inhibiting Interleukin 2 Production Angela M. Thornton and Ethan M. Shevach J. Exp. Med :

12. Cellular origin of CD4+ regulatory T cells
CD4+ Treg ?
Endogenous
Naturally-occurring
Induced during
Immune response

13. TReg cells are a normal product of thymic selection.
Figure 1. TR cells may arise from relatively high-avidity interactions with self-peptide–MHC complexes, just below the threshold for negative selection (green area). This narrow avidity selection window ensures that Tr cells will constitute only a small fraction of the mature T cell pool and have a greater sensitivity to self-peptide–MHC than potentially pathogenic autoreactive T cells.
Thymic development and TCR specificity: Unknown
Diverse T cell repertoire
Self-specific
Cross-reactive to foreign?
Altered negative selection?

14. Unique signals for CD4+CD25+ Treg cell homeostasis.
MHC II engagement in periphery
Foxp3
Requirement for co-stimulation:
CD40/CD40L
B7/CD28
IL-2 production
IL-2-/- mice
IL-2R signaling : IL-2Ra, STAT5, IL-2gc
Functional correlate: survival, mechanism…?
Other signals: TGF-b1

15. 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.

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

17. Hypothetical pathway to suppression.
Self-reactive
Teff cell √
Ag/TCR
nTreg
IL-2 transcription
CD4+CD25+
IL-2 protein
IL-2 driven
expansion
T cell differentiation &
effector function

18. Antigen Presenting Cell
CD4+CD25+ mediated suppression
is contact-dependent.
Requirement for APC?
CD4+
CD25+
T cell
Effector B
Pathways to
T cell suppression A X
Antigen Presenting Cell
Co-stimulation
Adhesion
CD4+CD25+ cells modulate co-stimulation?
MHC class I/II, B7.1/2, CD40 expression is unaffected.
Suppression is still operative with fixed LPS blasts.
Not overcome with  numbers of APCs.
Piccirillo et al. J.Immunology 167:

19. 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
Cytokine independent
Suppress IL-2 mRNA in T cells.
Suppression of effector functions
proliferation
inflammatory cytokines
differentiation
Effector molecules are unknown.
nTreg
Teff cell
Antigen Presenting Cell
CD4+CD25+
Suppressor Synapse
APC
CD4+
CD25+
CD4+
Teff

20. In vitro and in vivo role of cytokines in
CD4+CD25+ regulatory T cell-mediated suppression?
CD4+
CD25+
Effector
T cell
Cytokines ?
IL-4, IL-10
Immunosuppressive effects on APC and T cells
Suppression is cytokine independent
Cytokine neutralization
Absence of cytokines in suppressor supernatants
Cytokine-deficient Treg cells
Transwell chamber experiments
Contribution of
Transforming Growth Factor 1 (TGF-1) ?
Piccirillo et al. J.Exp. Med. 196:

21. 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 TGF-1?
Suppressor T cell-derived IL-10 ?
Nakamura et al JEM 2001
Membrane-bound TGF
Simon Read et al. JEM 2000.

22. Transforming Growth Factor 1
Requirement for
Transforming Growth Factor 1
(TGF-1) ?
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:

23. 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

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

25. 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-/-

26. 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

27. 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

28. Tissue-specific CD4+CD25+ mediated disease protection in the absence of IL-10.
IL-10-/- CD4+CD25+
CD4+CD25-
Nude
Gastritis
IBD
No Gastritis
IBD develops ! No
Gastritis
IBD

29. Diversity of modes of action
Context-dependent regulation of organ-specific autoimmunity.
Tissue-specific differentiation of Treg?
Any role for bacteria?
IBD is a bacterially-driven disease, not gastritis.
Lessons from germ-free mice.
Genetic background
Possible subsets of CD4+CD25+ Treg:
Cytokine versus Contact
Adaptable to inflammatory milieu.
Induction of other Treg cells.

30. Re-evaluation of CTLA-4 autoimmune phenotype
Could CTLA-4 deficiency result in defective Treg function and thus autoimmunity ? (rather than role of CTLA-4 on T cell autonomous regulation)
CTLA-4 phenotype rescued by complementing CTLA-4-/- BM with wild type BM (mixed BM chimera)
CTLA-4 deficient bone marrow can make CD25+CD4+ (function vs development)
CTLA-4 is expressed constitutively on CD4+CD25+CD45RBlow cells. Read et al. J. Exp. Med. July 2000

31. CD4+CD25+ (CD45low) inhibit IBD Anti–CTLA-4 treatment abrogates the Treg function.
Open: control Ig
Closed: CTLA-4.
Adoptive transfer of CD4 T cell into scid mice.

32. Possible Mechanisms of Action of CD4+ Regulatory T Cells
Naturally-occurring Treg
CD25
IL-10
TGF-1
Cellular
contact A
A+B B
MHC:
peptide
Activated
effector T cells
TCR
Naive
CD4+ or CD8+
T cells
APC
Tolerance
Autoimmunity
Transplantation
Tumor Immunity
Infectious disease
IL-10
TGF-1
Induced Treg
CD25