Regulatory T cells
Control of potential T cell self-reactivity: tolerance
Great opportunity to recognize every
pathogenic antigen
Random generation of an
immense T-cell repertoire
(~1015 different TCRs):
BUT
Potential self-reactivity
TOLERANCE:
• Central
5%
95%
• Peripheral
Tolerization mechanisms
T-cell INTRINSIC
Clonal
deletion
(apoptosis)
Anergy
T-cell ESTRINSIC
Ignorance
Regulatory T
cells
Regulatory T cells
Regulatory T cells (Tregs) represent a population of T cells that are
specialized for the suppression of the immune response;
Treg cells are essential for:
 Maintaining peripheral tolerance (preventing autoimmunity)
 Limiting chronic inflammatory diseases (immune homeostasis)
However, they:
 Limit beneficial immunity
 Limit antitumor immunity
Regulatory T cells: an old story…
Early 1970s: T cell-mediated suppression
however:
- lack of markers to distinguish suppressor T cells
- difficulties in isolating suppressor T cells for further studies
- lack of robustness of some suppressive phenomena
Regulatory T cells: re-discovered recently…
1995: S. Sakaguchi - Regulatory T cells
Regulatory T cells subsets
 CD4+CD25+Foxp3+ Treg
 Tr1
 Th3
 Other minor subsets (CD8+, CD4-CD8-,  T cells)
CD4+CD25+ Treg: origin
 thymus-derived (Natural occurring Treg or nTreg)
 induced in periphery ( CONVERSION)
Tcell
APC
Medium
affinity
High
affinity
Positive
selection
Apoptosis/
Tregs
% Foxp3
CD4+CD25+ Tregs
- constitute 10% of circulating CD4+ pool in normal mice
- they are anergic
- once activated, they do not produce IL-2
- they proliferate less than effector T cells in vitro after ag stimulation, but in
vivo they are continuosly proliferating due to self-ag recognition
- they consume IL-2
- they suppress effector function of other T cells
- once activated in an ag-specific manner, their suppression is not limited
to T cells with the same specificity
CD4+CD25+Foxp3+ Treg generation in the thymus
FoxP3 is induced in thymic
precursor cells upon engagement
with high-affinity TCR and other
costimulatory factors resulting in
FOXP3+ Treg cells. Different
functions associated with Treg cell
differentiation and function are
shown in the boxes.
Generation of Treg in the thymus:
- strong TCR engagement
- signals to common -chain containing cytokyne receptors
- CD28 costimulatory signals
CD4+CD25+ Treg: markers
CD25 (IL-2 receptor -chain)
 transiently expressed in activated T cells;
constitutively expressed in Tcells with regulatory abilitites
 at the steady state, 10% of peripheral CD4+ cells express CD25
“…when CD4+ cell suspensions prepared from normal mice are depleted of
CD25+ cells and are then inoculated into athymic nude mice, all recipients
spontaneously developed autoimmune disease…”
(S. Sakaguchi et al., 1995)
CD25+ cell
depletion
BALB/C
mouse
CD4+ cells
CD4+CD25cells
Athimic
Nude
mouse
“…when CD4+ cell suspensions prepared from normal mice are depleted of CD25+ cells and are then
inoculated into athymic nude mice, all recipients spontaneously developed autoimmune disease…”
(S. Sakaguchi et al., 1995)
Depletion of CD25+ cells is sufficient to eliminate T cells with
regulatory activity (Treg)
Autoimmunity
“CD4+CD25+ cells contribute to maintaining self-tolerance by downregulating immune response”
Foxp3 (Forkhead box P3)
• is located on the X chromosome
• forkhead/winged-helix transcription factor member
• is exclusively expressed on Tregs (vs. CD25):
all the T cells which express high levels of Foxp3 are Treg
BUT
Foxp3 is not expressed in every Treg
• its function is fundamental in Treg differentiation
Foxp3: IPEX and scurfy mice
IPEX (immune dysregulation, polyendocrinopathy,
enteropathy, X-linked)
• fatal autoimmune disease
• rare
• X-linked (as Foxp3 gene location!)
• characterized by Foxp3 mutation and hyperresponsive CD4+ cells
Scurfy mice
The scurfy mutation can be rescued by a transgene encoding Foxp3 allele
Foxp3 in Treg development and function
Foxp3 KO mice have lower
numbers of CD4+CD25+ cells
Expression of a Tg encoding Foxp3
convert naive T cells to Treg-like:
- confers suppressor ability
- CD25/CTLA4/GITR expression
- repress IFN-IL-4/IL-2 production
Conditional deletion of
Foxp3 in mature peripheral
Tregs results in
- loss of suppressor function
- production of IL-2 and other
pro-inflammatory cytokines
Control of Treg function by Foxp3
Interaction with TFs
Control of Treg function by Foxp3
Target genes
Foxp3 controls directly or indirectly nearly 700 genes
Foxp3 binds directly to nearly 10% of them
Among target genes:
• signal transduction genes
• transcription factors (!!!!!!!)
• cytokines (e.g.Il2)
• cell surace molecules
• enzymes for cell metabolism
• miRNA
Foxp3 functions as an activator as well as a repressor of the
transcription depending on the target
CTLA-4 (Cytotoxic T lymphocyte antigen)
• CD28-family receptor
• It binds the same ligands as CD28 (CD80 and CD86)
• higher affinity than CD28
• T cell inibitory receptor CTLA4 KO die
prematurely for multiorgan infalmmation
In nTregs:
• Constitutively expressed (vs.
transient expression in other T)
• CTLA4 expression is controlled
by Foxp3
• required for their in vivo and in
vitro suppression, (down-regulation
of CD80 and CD86 on APC)
GITR (glucocorticoid-induced tumor
necrosis factor receptor)
•Expressed in activated T cells or in Tregs
•role of GITR in attenuating the suppressive activity of CD4+ CD25+ T cells
IL-2 and Tregs
Mechanisms of suppression
Mechanisms of suppression: inhibitory cytokines
TGF- and IL-10:
IL-35:
-fundamental for iTreg mediated
suppression
-Their contribution to nTreg
mediated suppression is debated
-Recently discovered in mice
-Not produced by human Tregs
Mechanisms of suppression: cytotoxicity
Cell-contact dependent mechanism
Mechanisms of suppression: metabolic disruption
Cell-contact dependent
mechanism (or close
proximity)
Competition for cytokines (es IL-2 deprivation)
Delivery of a negative signal to Teff:
- upregulation of cAMP T cell proliferation and IL-2 production
- generation of pericellular adenosine T cell function
Mechanisms of suppression: functional modification of APC
Repression of APC function/
maturation:
-  CD80/CD86 expression via
CTLA4
- indoleamine 2,3-dioxygenase
(IDO) production
- LAG-3 dependent block of
maturation
Relative contribution of different mechanisms of suppression
Hyp 1: operate synergistically and sequentially
Hyp 2: different mechanism for different scenario (contextual model)
Hyp 3: one/few critial and many accessory mechanism (hierarchical model)
Induced or adaptive regulatory T cells (iTregs)
• Foxp3+ iTregs
• Tr1
• Th3
Role of Treg in infectious disease
• limit the magnitude of effector response (advantage/disadvantage for the
host)
• limit collateral tissue damage (advantage)
Infection
activates both
Tregs and
effector
function
Treg
function
Effector
response
Outcome
of an
infection
Nature Immunology 6, 353 - 360 (2005)
Role of Tregs in cancer
preventing autoimmunity
Treg suppress host immune response
Limit anti-tumor immunity
Ovarian tumors are infiltrated by
both effector (CD4+CD25-) T
cells and by an excess of TREG
cells (CD4+CD25+). These
TREG cells impede the function
of the effectors in combating the
tumor. The chemokine CCL22,
produced by the tumor cells,
binds to its receptor (CCR4) on
the TREG cells and mediates
their recruitment into the tumor.