Regulatory Lymphocytes of the Immune System'

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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
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Induced regulatory T cells
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. 2004.
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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 lt-gt 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

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B. NATURALLY-OCCURRING Endogenous in naïve
repertoire professional
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
_
_
Activated Effector T cell
Autoimmunity Transplantation Tumor Immunity
Infectious disease
Piccirillo et al. Trends in Immunol. 2004.
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Journal of Immunology 1995 1551151
Lymph nodes from a 2 month old BALB/c
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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
CD4CD25 Treg cells
Tissue regeneration
Mucosal Immunity
Transplantation

• Gastritis
• Oophritis
• Orchitis
• Thyroiditis
• Pancreatitis
• Colitis
• Auto Abs

• Depletion
• Day3 thymectomy
• Genetic anomalies
• Irradiation
• Infections
• Drugs

Normal Immune responses
Tumor Immunity
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CD4CD25 nTreg cellsMasterswitch of peripheral
tolerance
Immunity
Tolerance
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Reliable biomarkers for CD4CD25 regulatory T
cells?
CD25
GITR
CTLA-4
CD4CD25 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
Unidentified X,Y,Z ??
Neuropilin-1
OX-40
Piccirillo et al. Trends in Immunology July
2004
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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.
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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 (?)

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CD4CD25 regulatory T cells suppress T cell
functions. Consensus elements
CD8
24h
Cytokine Production
Proliferation
CD8/-
CD8/
CD4 or CD8 T cells
CPM
48h
CD4CD25- ( ? ) or CD4CD25 (? ) 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/
CD4CD25 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.
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Cellular origin of CD4 regulatory T cells
CD4 Treg
?
Endogenous Naturally-occurring
Induced during Immune response
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TReg cells are a normal product of thymic
selection.
Figure 1. TR cells may arise from relatively
high-avidity interactions with self-peptideMHC
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-peptideMHC 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?

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Unique signals for CD4CD25 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

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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.

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Fontenot et al.
More selective and faithful marker than CD25
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Hypothetical pathway to suppression.
v
Self-reactive Teff cell
Ag/TCR
nTreg
IL-2 transcription
CD4CD25
IL-2 protein
IL-2 driven expansion
T cell differentiation effector function
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CD4CD25 mediated suppression is
contact-dependent. Requirement for APC?
T cell Effector
CD4 CD25
B
Pathways to T cell suppression
X
A
Antigen Presenting Cell Co-stimulation Adhesion

• CD4CD25 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 1671137-1140.
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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
• Cytokine independent
• Suppress IL-2 mRNA in T cells.
• Suppression of effector functions
• proliferation
• inflammatory cytokines
• differentiation

nTreg
Teff cell
Antigen Presenting Cell
CD4CD25
Suppressor Synapse
APC
CD4 Teff
CD4 CD25
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In vitro and in vivo role of cytokines in
CD4CD25 regulatory T cell-mediated
suppression?
Effector T cell
CD4 CD25
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. 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 TGF-?1? Suppressor
T cell-derived IL-10 ?
Nakamura et al JEM 2001 Membrane-bound TGF
Simon Read et al. JEM 2000.
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• Requirement for
• Transforming Growth Factor ?1
• (TGF-?1) ?

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-/-
A
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 ()
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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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Tissue-specific CD4CD25 mediated disease
protection in the absence of IL-10.
CD4CD25- CD4CD25
CD4CD25- IL-10-/- CD4CD25
CD4CD25-
Nude
Gastritis IBD
No Gastritis IBD develops !
No Gastritis No IBD
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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 CD4CD25 Treg
• Cytokine versus Contact
• Adaptable to inflammatory milieu.
• Induction of other Treg cells.

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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 CD25CD4
  (function vs development)

CTLA-4 is expressed constitutively on
CD4CD25CD45RBlow cells. Read et al. J. Exp.
Med. July 2000
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CD4CD25 (CD45low) inhibit IBD AntiCTLA-4
treatment abrogates the Treg function.
Open control Ig Closed CTLA-4. Adoptive
transfer of CD4 T cell into scid mice.
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Possible Mechanisms of Action of CD4 Regulatory
T Cells
Naturally-occurring Treg
CD25
IL-10 TGF-?1
Cellular contact
A
B
AB
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