REGULATORY CD4 T CELLS

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REGULATORY CD4 T CELLS
KEVIN MALOY Sir William Dunn School of
Pathology University of Oxford South Parks Road
kevin.maloy_at_path.ox.ac.uk
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HISTORICAL PERSPECTIVE

• 1970s - existence of specialized suppressor T
  cells postulated
• - usually attributed to Lyt2 (CD8) TS cells
• - variety of suppressive phenomena and networks
• 1980s - advent of molecular immunology
• - inability to identify cellular and molecular
  mechs of suppression
• - stigma associated with TS cells
• 1990s - phenotypic identification of CD4 T cell
  subsets
• - distinct patterns of cytokine secretion Th1,
  Th2 etc.
• - immunomodulatory roles for cytokines
• 1994-present - rebirth of suppressor T cells as
  regulatory TR cells
• - phenotypic and functional characterization of
  TR cells
• - role in preventing autoimmunity and immune
  pathology

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Evidence for naturally occurring TR cells
Experimental models of autoimmunity -
organ-specific autoimmunity models in mice
(Sakaguchi, Shevach) - diabetes, thyroiditis
models in rats (Mason, Penhale) Experimental
models of T-cell mediated immunopathology -
inflammatory bowel disease (IBD) model
(Powrie) - TCR tg EAE model (Lafaille) Common
characteristics of these models - disease
involves manipulation of T cell homeostasis -
disease inhibited by a subpopulation of normal
CD4 T cells
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T cell adoptive transfer model of inflammatory
bowel disease
Normal mouse CD4 T cells
(Naïve)
(Activated/Memory)
Immunodeficient mouse (scid or RAG-/-)
COLITIS
NO DISEASE
Th1 response IFNg/TNFa
IL-10/TGF-b
Powrie et. al.
5
CD45RBlow CD4 cells can be subdivided by
expression of CD25
CD25 CD45RBlow
CD25
CD45RB
CD25- CD45RBlow
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TR cells which inhibit colitis are enriched
within the CD25CD45RBlow population
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3
Colitis Score
2
1
0
2x105 5x104
CD45RBhigh alone CD45RBhigh CD45RBlow
CD45RBhigh CD25RBlow CD45RBhigh CD25-RBlow
Read et. al. JEM (2001) 192 295.
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Experimental models of autoimmunity provide
further evidence of the existence of CD25CD4 TR
cells
Ablation of TR cells causes organ-specific
autoimmunity in mice
A)
Gastritis Oophoritis Orchitis Thyroiditis
X
CD25-CD4 T cells
B)
T cell deficient
CD25CD4 T cells from normal mice
Sakaguchi et. al.
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Naturally occurring CD4 TR cells
Present in the T cell repertoire of normal
animals (rodents and humans)
Suppress harmful immunopathological
responses directed against self or foreign
antigens
Reside mainly within the subpopulation of
CD25CD4 T cells (N.B. CD25-CD4 T cells also
have some regulatory potential)
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OTHER POPULATIONS OF CD4 TR CELLS
Levings et. al. JI (2001) 1665530.
Vendetti et. al. JI (2000) 1651175.
Weiner, H.L. Nat. Immunol. (2001)
Hammond Kronenberg Curr. Opin. Immunol. (2003)
Relationships between these TR populations and
naturally occurring CD25 CD4 TR?
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Naturally occurring CD25 CD4 TR cells
Phenotypic characteristics

• Constitute 10 of peripheral CD4 T cells
• Also found in the thymus (5 of CD48-SP
  thymocytes)
• Present in mice, rats and HUMANS
• 4. Partially anergicin vitro
• - proliferate poorly upon TCR ligation
• - dont produce IL-2
• Activated/memory phenotype CD25CD45RBlowCD44hi
• Constitutively express high levels of GITR and
• high proportion (50) express CTLA-4

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CD25 is not an absolute marker for TR cells

• CD25 CD4 T cell population is heterogenous
• - CD25 transiently expressed by activated CD4
  T cells
• - CD25 expression is highly dynamic in vivo
• CD25- CD4 T cell populations have some TR cell
  activity
• - usually less potent than CD25 CD4 T cells
• - prevent EAE in MBP-specific TCR tg mice
  (Lafaille)
• - can inhibit diabetes and IBD in rodent models

Not all CD25CD4 T cells are TR cells Not all TR
cells express CD25
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CD25 CD4 TR cells inhibit the proliferative
responses of other T cells in vitro
T cells cultured
Stimulus
Proliferation

CD25-CD4
a-CD3 APC
-
CD25CD4
a-CD3 APC

-
a-CD3 APC
Shevach et. al.
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In vitro suppression by CD4 CD25 TR is
cell-contact dependent
Transwell culture system
a-CD3
CD4 CD25
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In vitro suppression by CD4 CD25 TR is
cell-contact dependent
CPM x 10-3
Does not involve Killing of responder T
cells Cytokine secretion by TR (TGF-b??)
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Naturally occurring CD25 CD4 TR cells in vivo
regulatory functions

• Inhibit a number of experimental autoimmune
  diseases
• in rodent models - gastritis, thyroiditis,
  diabetes, EAE
• Prevent IBD in murine models
• Mediate tolerance to allografts
• Impede anti-tumor immunity
• Suppress other T cell responses (CD4 T and CD8)
  
• Inhibit pathological immune responses
• mediated by innate or adaptive immune systems
• 7. Regulate responses against infectious agents

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Mechanisms involved in the suppression of immune
responses by CD25 CD4 TR cells

• Still not completely understood
• Difficult to define precise cellular/molecular
  pathways in vivo
• Mechanisms may vary depending on the experimental
  model
• local environmental factors in target organ
• nature of immunopathological response

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Cytokines play a key role in the control of
intestinal inflammation by TR cells
X
colitis
CTLA-4
CD4 CD45RBlow CD25
Inhibition of colitis is dependent on IL-10,
TGF-b and CTLA-4 (a-IL-10R or a-TGF-b or a-CTLA-4
all abrogate suppression)
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Surface molecules and TR cells
CTLA-4 and GITR have been functionally implicated
in suppression in vivo
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Development of CD25 CD4 TR cells
Are TR cells imprinted with this function in the
thymus? or Do TR cells develop from naïve T cells
in the periphery?
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Thymic generation of TR cells
CD4 SP thymocytes are a potent source of TR
cells - 5-10 express CD25 in humans, rats,
mice CD25CD4 thymocytes have similar
suppressive activities to peripheral CD25 TR
cells - inhibit gastritis, diabetes, colitis in
vivo - inhibit T cell proliferation in vitro
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Development of CD4CD25 TR in the thymus
Jordan et al. (2001) Nat Immunol. 2301.
50 of CD4 LN cells are CD25 TR CD25 cells in
thymus of 2d old mice - not due to
recirculation of peripheral cells - selected
on radioresistant thymic epithelium Remaining
50 of CD25- cells are responsive to antigenic
stimulation
Double tg mice
X
High affinity TCR tg
HA Tg (thymus periphery)
X
Normal low levels of CD25 cells
Low affinity TCR tg
CD4CD25 TR generated by high affinity
interactions with self-peptide presented on
radioresistant thymic epithelial cells
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TR cells may arise from relatively high avidity
interactions with self-peptide-MHC complexes
Probability of selection
Avidity
Treg
negative selection
neglect
positive selection
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Transcription factor Foxp3 controls TR cell
development
Hori et. al. Science (2003) 2991057.
Foxp3 is predominantly expressed by CD4CD25 T
cells in both thymus and periphery
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Foxp3-transduced naïve T cells show TR cell
activity
Retroviral transduction of Foxp3 into naïve
CD4CD25- T cells enables them to protect against
colitis and gastritis in vivo
Humans with mutations in Foxp3 develop
multi-organ autoimmune diseases, allergy and IBD
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Specificity of TR cells?
Little known about the peptide ligands recognized
by TR cells

• Once activated TR effector function is
  non-Ag-specific
• can inhibit the responses of T cells recognizing
  different Ags
• bystander suppression

Available evidence suggests TR cells are
heterogenous population that can react with
peptides derived from both self-tissues and
foreign antigens
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Acquisition of TR cell function in the periphery

• Transplantation models. (Waldmann/Cobbold)
• - CD4 TR cells from tolerant mice can transfer
  this tolerance and can educate naïve T cells to
  develop into TR cells in the periphery,
  infectious tolerance.
• Tolerogenic antigen application can prime TR
  cells.
• - mucosal Ag delivery (orally or i.n.)
• - soluble peptide i.v.
• 3. Cytokines and APC.
• - IL-10 and TGF-b
• - immature DC

promote TR cell differentiation in vitro
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Naturally-occurring versus inducible TR cells
Periphery
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TR cells and the equilibrium between protective
immunity and immune pathology
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Pathogen-activated DC may drive TR cell
development
Mills K.H.G. Nat. Rev. Immunol. (2004) 4841
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• P. carinii
• opportunistic pulmonary pathogen
• resistance mediated by CD4 T cells
• excessive immune activation may cause pneumonia
  (PCP)
• mainly in immunocompromised hosts

Monitor Lung inflammation T cell
infiltration Pathogen eradication
RAG-/-
P.carinii
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CD25- cells alone Lethal pneumonia T cell
infiltration
CD25- CD25 cells Survival - minimal
inflammation Reduced T cell infiltration
But CD25 cells also prevent eradication of
P.carinii
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L.major infection instrumental in discovery of
CD4Th cell subsets BALB/c mice ? Th2 resp ?
disseminated disease ? B6 mice ? Th1
resp ? survival
B6 mice maintain a few viable organisms in
original lesion, but are resistant to secondary
challenge at another site (Concomitant immunity)
How is this paradoxical situation maintained?
Aim To test the hypothesis that CD4CD25 TR
cells allow L.major parasites to persist in
resistant mice in order to maintain concomitant
immunity
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CD4CD25 TR cells persist at the lesion site in
L.major-infected B6 mice
CD4CD25 T cells from lesion - inhibit prolif
and IFN-g secretion of CD4CD25- lesion T
cells - secrete IL-10 in response to L.major
Ags - suppress immunity to L.major mediated by
CD4CD25- T cells
CD4CD25 TR cells allow a few L.major to persist
in the 1 lesion Host cost disease spread and
potential for re-activation Host benefit
maintains effective immunity against re-infection
Equilibrium develops between TE and TR cells to
regulate pathogen immunity and minimise host
tissue damage
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Modulation of TR cell activity by inflammatory
stimuli - TR activity beneficial against
autoimmunity but may impede effective
anti-pathogen responses - Are additional
mechanisms activated during infection that could
abrogate the effects of TR cells?
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DC activation and CD25 TR cell suppression in
vitro
CD4 T cells cultured
Stimulus
Proliferation
a-CD3
Y

Y
Y
Y
Y
Y
Y
Y
-
Does microbial activation of DC overcome TR cell
suppression?
Test by adding LPS or CpG to suppression assays
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Ligation of TLRs on DC overcomes TR cell
suppression
Activated DC overcome TR cell activity by
secreting IL-6 and other soluble factors
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Therapeutic potential of TR cells
Increase TR Autoimmune Inflammatory
disease Transplantation
Decrease TR Cancer Infectious disease
Caution required - delicate balance between
enhancing IRs while avoiding autoimmunity
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Can TR cells ameliorate established colitis?
Sacrifice some at 4 weeks to determine colitis
score
CD4 CD45RBhigh
Score colitis at 8-14 weeks
CB-17 scid
CD4 CD25
Add CD25 cells at 4 weeks
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Cure of chronic colitis by TR cells
Mottet, Uhlig, Powrie. JI (2003) 170 3939.
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TR cells migrate to inflammatory sites
TR cells identified in many inflammatory
lesions - Intestinal lamina propria - L. major
skin lesions - P.carinii lungs - Tolerated
allografts
How do they get there? - Adhesion molecules? -
Chemokine receptors Human CD4CD25 T cells
express CCR4 CCR8 CCL4 attracts CD4CD25 T
cells (mice)
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A) Steady State LN
C) Regulatory phase
IL-10
B) Response phase LN
IL-10
TLR

Tmem
LN
GITR-L CD28
Inflammatory Site
IL-6
IL-10
IL-2
TGF-b
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Further reading

• Maloy, K.J. and Powrie, F. Regulatory T cells in
  the control of immune pathology. Nat. Immunol.
  (2001) 2816.
• Shevach, E.M. CD4CD25 suppressor T cells more
  questions than answers. Nat. Rev. Immunol. (2002)
  2389.
• Sakaguchi, S. Naturally arising CD4 regulatory T
  cells for immunologic self-tolerance and negative
  control of immune responses. Annu. Rev. Immunol.
  (2004) 22531.
• Mills, K.H.G. Regulatory T cells Friend or foe
  in immunity to infection? Nat. Rev. Immunol.
  (2004) 4841.