View
218
Download
3
Category
Tags:
Preview:
Citation preview
Regulatory Lymphocytes of the Immune System.
Dr. C. PiccirilloCanada Research Chair
Department of Microbiology & ImmunologyMcGill University
MIMM-414ALecture 2- Oct. 23, 2006
ActivatedEffector
T cell
APC
_
+ Peripheraldifferentiation
signals
Thymic CD4Thymic CD4++ T cell pool T cell pool
Peripherally-induced CD4+ Treg cells
( iTreg )
Autoimmunity Transplantation Tumor Immunity
Infectious disease
TCR
Piccirillo et al. Trends in Immunol. 2004.
Induced regulatory 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
• A. INDUCED - immunization, experimental manipulation…
– NK-T cells– CD8+ T cells T cells– DN T cells
A network of CD4+ regulatory T cellscontrol immune reponses.
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.
B. NATURALLY-OCCURRINGEndogenous in naïve repertoire: professional
Journal of Immunology 1995 155:1151
Lymph nodes from a 2 month old BALB/c
Normal Immune
responses
Tissue regeneration
Allergy
Tumor Immunity
Transplantation
Infectious disease
Autoimmunity
CD4+CD25+ Treg cells
MucosalImmunity
1 10 100 1000 100001
10
100
1000
100009
FL2-H: CD25 PE
FL
3-H
: C
D4
Tri
Colo
r
• Depletion• Day3 thymectomy• Genetic anomalies• Irradiation• Infections• Drugs
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
Naturally-occurring CD4+ regulatory T cells.
Gastritis Oophritis Orchitis Thyroiditis Pancreatitis ColitisAuto Abs
CD4CD4++CD25CD25++ nTreg cells nTreg cellsMasterswitch of peripheral toleranceMasterswitch of peripheral tolerance
Reliable biomarkers for CD4+CD25+ regulatory T cells?
CD25
CTLA-4GITR
CD103 Galectin-1
Ly6
OX-40
4-1BB
Unidentified X,Y,Z ??
Neuropilin-1
CD4+CD25+
RegulatoryT cell
Piccirillo et al. Trends in Immunology July 2004
NONO marker after T cell activation.
OnlyOnly in the naïve T cell repertoire.
Need better marker.
MHC/peptide
TCR
Activated CD4+ T cell
RestingCD4+ T cell
T cell activation induces expression of functional T cell
surface molecules
TCR
APC
CD40L
CD25CD69CD44
CD25 is not a marker of Treg after T cell activation.Only in the naïve T cell repertoire.
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 (), TGF1 ()
Phenotypic and functional characteristics
CD4+CD25+ regulatory T cells suppress T cell functions. Consensus elements
IFN
- (
ng
/ml)
0
4
8
12
16
20
24
28
CD8+ CD8+/CD4+CD25-CD8+/CD4+CD25+
Cytokine Production
IFN-
CD8+ CD8+/- CD8+/+
24h
48h
CD8+
CD8+/-
CD8+/+
CP
M
# CD4+CD25- ( ) or CD4+CD25+ ( ) T cells
0
25000
50000
75000
100000
125000
0 10000 20000 30000 40000 50000
CD4+ or CD8+
T cells
Proliferation
• Requirement for TCR engagement• Antigen non-specific & MHC unrestricted
• Induce cell cycle arrest (G1-S transition)
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. 1998 188: 287-296.
Cellular origin of CD4+ regulatory T cells
CD4+ Treg
EndogenousNaturally-occurring
Induced duringImmune response
?
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.
TReg cells are a normal product of thymic selection.
Thymic development and TCR specificity: Unknown
Diverse T cell repertoire
Self-specific
Cross-reactive to foreign?
Altered negative selection?
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-2R, STAT5, IL-2c Functional correlate: survival, mechanism…? Other signals: TGF-1
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
Hypothetical pathway to suppression.
Ag/TCR
IL-2 transcription
IL-2 protein
IL-2 driven expansion
T cell differentiation & effector function
Self-reactiveTeff cell
nTreg
CD4+CD25+
√
T cellEffector
CD4+
CD25+
Pathways to T cell suppression
Antigen Presenting CellCo-stimulation
Adhesion
XA
B
• 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.
CD4+CD25+ mediated suppression is contact-dependent.
Requirement for APC?
Piccirillo et al. J.Immunology 167:1137-1140.
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
• Cytokine independent
• Suppress IL-2 mRNA in T cells.
• Suppression of effector functions
• proliferation• inflammatory cytokines• differentiation
• Effector molecules are unknown.
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) ?
Piccirillo et al. J.Exp. Med. 196:237-250.
EffectorT cell
CD4+
CD25+
Cytokines ?
In vitro and in vivo role of cytokines in CD4+CD25+ regulatory T cell-mediated suppression?
• 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
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 TGF-1?
Suppressor T cell-derived IL-10 ?
Initial studies showed that anti-IL-10 or anti-TGF-B1abrogated Treg-mediated suppression of disease.
Nakamura et al JEM 2001Membrane-bound TGFSimon Read et al. JEM 2000.
Requirement for Transforming Growth Factor Transforming Growth Factor 1 1
(TGF-(TGF-1) 1) ?
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?
100806040200
TGF-b KO CD25pos
WT CD25neg + TGF-b KO CD25pos
WT CD25neg + WT CD25pos
WT CD25neg
No cells
Severe colitis (3.5 - 4.0)Moderate colitis (2.0 - 3.0)Mild colitis (1.0 - 1.5)No colitis (0 - 0.5)
Incidence of colitis (%)
Colon grades (HEL129 + HEL141)
n=8
n=7
n=8
n=8
n=9
CD25– CD25+
cells cells
— — WT —
WT WT
WT TGF-1-/-
— TGF-1-/-
A
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
Incidence of colitis (%)
B
TGF-1-/- CD4+CD25+ nTreg cells suppress IBD.
CD25– CD25+ cells cells
— —
WT —
WT WT
WT TGF-1-/-
— TGF-1-/-
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?
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 !
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.
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
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.
MHC:peptide
NaiveCD4+ or CD8+
T cells
Possible Mechanisms of Action of CD4+ Regulatory T Cells
TCR
APC
CD25
Activated effector T cells
CD25
Naturally-occurring Treg
Induced Treg
IL-10TGF-1
IL-10TGF-1
Cellularcontact
A BA+B
Tolerance Autoimmunity
Transplantation Tumor Immunity
Infectious disease
Recommended