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PERIPHERAL TOLERANCE
REGULATORY T-CELLS
ARPAD LANYI PhD
SELF/NON-SELF DISCRIMINATION
is a central issue in immunology
Immunological homeostasis
TOLERANCE
IMMUNITY
IMMUNODEFICIENCIES
PERSISTENT INFECTIONS
TUMOURIGENESIS
AUTOIMMUNE DISEASES
CHRONIC INFLAMMATION
REGULATORS
EFFECTORS
SELF/NON-SELF DISCRIMINATION
is a central issue in immunology
Key Questions:
What goes wrong in autoimmune disease?
What should be improved to treat autoimmune disease?
What should be strengthened to maintain active self tolerance to transplants?
What is the role of regulatory cells in normal immune responses?
Allergic reactions?
To answer these questions we need to learn the biology of Tregs!!!
CENTRAL TOLERANCE
PERIPHERAL TOLERANCE
Exposure of CD4+T-cells to an antigen in the
absence of co-stimulation or innate immunity
may make the cells incapable of responding
to that antigen.
Regulatory T lymphocytes are a subset of
CD4+ T-cells whose function is to suppress
immune responses and maintain selftolerance.
T-cells that recognize self antigens with
high affinity or are repeatedly stimulated by
antigens may die by apoptosis.
REGULATORY T-CELLS
NOMENCLATURE
Investigation of regulatory T-cells is a hot topic in immunology. Several types of
Treg populations have been identified: for example CD4+ IL-10-producing Tr1 cells,
TGF-β producing Th3 cells, CD8+CD28-T-cells, HLA-E-specific CD8+T-cells etc. This
lecture is about the best characterized CD4+ CD25+FOXP3+ Tregs.
In the absence of standard nomenclature, in this lecture the following terms and
abbreviations are going to be used (other terms are in brackets):
 Treg: FOXP3+ regulatory T-cells in general
 Thymic regulatory T-cells/tTreg (naturally occuring/nTreg)
 Peripheral regulatory T-cells/pTreg (adaptive/induced/a/iTreg)
 In vitro generated regulatory T-cells/iTreg
EARLY EXPERIMENTAL MODELS SUGGESTING THE
PRESENCE OF REGULATORY T-CELLS
Thymic regulatory T-cells (Sakaguchi S.)
Function:
Maintenance of natural self-tolerance
Control of pathological as well as physiological immune responses
SELF-TOLERANCE:
Murine neonatal thymectomy at day 2-4
destruction of ovary (autoimmune)
Thymectomy + Xray in adult rat
autoimmune thyreoditis
Both phenotype reversed by injection of CD4+ T-cells suggests a suppressor population must exist.
• Self tolerance is dominant, and should be
maintained by a population of suppressor cells.
• Depletion of Treg cells? OK, but markers????
IDENTIFICATION OF THE TREG LINEAGE AND ITS CELL
SURFACE MARKERS BY DEPLETION EXPERIMENTS
Nude (athymic) mice (no T-cells)
+ transfer normal CD4+ T-cells
No autoimmunity
Nude (athymic) mice (no T-cells)
+ transfer CD5-depleted CD4+
T-cells (antibody + complement)
Autoimmunity
Treg
Teffector
Sakaguchi et al. showed in Ann rev immun. 2004
stomach, thyroid,
ovaries, testes
FOXP3: MORE THAN A LINEAGE MARKER
doi:10.1038/nri3650
Ectopic expression of FOXP3 in naive mouse CD4+ T-cells confers suppressive activity and induces the
expression of Treg-associated signature molecules such as CD25, CTLA4 and GITR. Expression of these
receptors also correlates with FOXP3 expression
in human CD4+ T-cells.
FOXP3 has a key role in maintenance of self tolerance.
FOXP3 DEFICIENCY:IPEX
Immune dysregulation, polyendocrinopathy, enteropathy, X-linked
syndrome
 Polyendocrinopathy: multiple disorders of the endocrine glands.
 Type 1 diabetes mellitus (most common) develops within the first few months of life.
 Autoimmune thyroid disease (hypo- or hyperthyroidism).
 Enteropathy: severe diarrhea, usually the first symptom, failure to thrive.
 Dermatitis.
pruriginous plaques (a)
eczema-prurigo (b)
extensive cutaneous
involvement with diffuse
psoriasiform plaques (c,d)
atopic dermatitis and severe
bleeding cheilitis (e)
DOI 10.1111/j.1365-2133.2008.08835.x
FOXP3 DEFICIENCY:IPEX
Immune dysregulation, polyendocrinopathy, enteropathy, X-linked
syndrome
 Polyendocrinopathy: multiple disorders of the endocrine glands.
 Type 1 diabetes mellitus (most common) develops within the first few months of life.
 Autoimmune thyroid disease (hypo- or hyperthyroidism)
 Enteropathy: severe diarrhea, usually the first symptom, failure to thrive.
 Dermatitis.
 Autoimmune blood disorders are common; about half of affected individuals have anemia,
thrombocytopenia, neutropenia.
 IPEX syndrome may involve the liver and kidneys (tubular nephropathy).
 Most patients with IPEX are males and most of them die within the first 2 years of life
without treatment (a few with a milder phenotype have survived into the second or third
decade of life).
 Treatment: hematopoietic stem cell transplantation (HSCT).
DIFFERENTIATION AND CHARACTERIZATION
OF
REGULATORY T-CELLS
TREG DIFFERENTIATION
THYMIC REGULATORY T-CELLS
(tTreg)
Requirements for tTreg development:
TCR signaling
High avidity self-specific TCR:
greater sensitivity to self-peptide–MHC than
potentially pathogenic autoreactive T-cells.
Co-stimulation
Decreases in frequencies of Tregs in
CD28-deficient and CD80-CD86-deficient
mice.
Cytokine-mediated signals
IL-2
TREG DIFFERENTIATION
Hassall’s corpuscles instruct dendritic cells
to induce CD4+CD25+ regulatory T-cells in
human thymus
Watanabe et al. Nature 436, 1181
doi:10.1038/nature03886
TREG DIFFERENTIATION
THYMIC REGULATORY T-CELLS
(tTreg)
Requirements for FOXP3 expression:
TCR signaling
High avidity self-specific TCR:
greater sensitivity to self-peptide–MHC than
potentially pathogenic autoreactive T-cells.
Co-stimulation
Decreases in frequencies of Tregs in
CD28-deficient and CD80-CD86-deficient
mice.
Cytokine-mediated signals
IL-2
Control of systemic and
tissue-specific autoimmunity
Ongoing TCR
engagement
TREG DIFFERENTIATION
PERIPHERAL REGULATORY
T-CELLS (pTreg)
Low level TCR signaling:
Suboptimal DC activation
Sub-immunogenic doses of agonist peptide
While CD28 signaling is critical for thymic
selection of Tregs, peripheral Treg induction is,
in contrast, inhibited by strong CD28 ligation.
Cytokine-mediated signals
IL-2, TGF-β
tissue-specific
or foreign
antigen
CD4+FOXP3+pTreg
FOXP3+pTreg
TREG DIFFERENTIATION
PARALLEL OR SEQUENTIAL DEVELOPMENT OF
EFFECTOR AND REGULATORY T-CELLS
The life of regulatory T-cells Iris K. Gratz, Michael D. Rosenblum, and Abul K.
Abbas Ann. N.Y. Acad. Sci. (2013) 1–5
Local immunosuppression
TREG DIFFERENTIATION
Mucosal, oral, fetal-maternal tolerance
PERIPHERAL REGULATORY
T-CELLS (pTreg)
Low level TCR signaling:
Suboptimal DC activation
Sub-immunogenic doses of agonist peptide
While CD28 signaling is critical for central
selection of Tregs, peripheral Treg induction is,
in contrast, inhibited by strong CD28 ligation.
Cytokine-mediated signals
IL-2, TGF-β
ATRA
Synergizes with TGF-β
Sufficient to allow pTreg development even
in the presence of high levels of costimulation.
tissue-specific
or foreign
antigen
FOXP3+pTreg
FOXP3+pTreg
FUNCTIONAL
PLASTICITY
Th1
IFN-γ
pTregs are able to adapt to the local environment
by transcriptional regulation and mediate CXCR3
suppression of the specific type of inflammation
T-bet and GATA3
double deficiency in
Tregs results in
autoimmunity.
Ablation of IRF4 in Tregs
results in autoimmune
lymphoproliferative
disease.
Peripheral T-regs can partially mimic the
phenotype of the target T-cells.
CXCR5
Tfh
Cxcr5-/-
Bcl6-/-Treg
Both
Treg and
are inefficient in controlling
germinal center reactions.
IL-4
IL-5
IL-13
CCR4
FOXP3
This functional plasticity seems to be essential
for suppression.
pTREG CELLS CAN EVEN
CONVERT INTO EFFECTOR T-CELLS!
STAT6
IRF4
GATA3
STAT1
Tbet
Th2
IL-21
IL-4
Bcl6
STAT3
RORγ
Treg-specific ablation
of Stat3 results in
the development of
fatal intestinal
inflammation.
CCR6
Th17
IL-17
FOXP3+ T-CELLS ARE HETEROGENEOUS
NOTE THAT IN HUMANS
THE EXPRESSION OF THE LINEAGE MARKER FOXP3 IS NOT EXCLUSIVE
Conventional T-cells transiently express FOXP3 at low level following activation
though it does not enable supression.
Three populations of FOXP3 expressing cells:
CD25+CD45RA-FOXP3lo (non-suppressive activated T-cells)
CD25+CD45RA+FOXP3lo (resting naive Treg)
CD25hiCD45RA-FOXP3hi (effectorTreg)
FOR PERSISTENT SUPPRESSIVE FUNCTION
STABLE AND HIGH FOXP3 EXPRESSION IS REQUIRED
MULTIPLE SIGNALING PATHWAYS
CONTRIBUTE TO ACTIVATION
AND MAINTENANCE OF
FOXP3 EXPRESSION
doi:10.1038/nri2474
EPIGENETIC CONTROL OF FOXP3 EXPRESSION
CNS2: TREG-SPECIFIC DEMETHYLATED REGION (TSDR)
MAINTENANCE OF STABIL FOXP3 EXPRESSION.
CNS3: NF-κB family members bind to this element and
CNS2-deficient mice: FOXP3 induction in the thymus and in the periphery
Classic TATA and
facilitate the activity of the FOXP3 promoter.
was
unimpaired
BUT
progressive
lost of FOXP3 expression upon division.
CAAT-box-containing promoter
CNS3 deficient mice remain healthy despite decrease
FOXP3 is recruited to CNS2.
in repertoire of Treg cells.
Partial demethylation
p
doi: 10.3389/fimmu.2013.00169
CNS1 (Conserved non-coding sequence):
TGF-β sensor
CNS1 deficient mice:
No detectable tissue-specific autoimmunity BUT
impaired pTreg generation.
Th2-type pathologies at mucosal sites.
Increased embryo resorption.
SUMMARY
CD4+CD25+FOXP3+ REGULATORY T-CELLS
THYMIC TREG
PERIPHERAL TREG
Self antigen specific TCR
Self or non-self antigen specific TCR
Stable FOXP3 expression
Functional plasticity
Control of systemic autoimmunity
Local immunosuppression
IN VITRO GENERATED REGULATORY T-CELLS
(iTREG)
Murine iTreg:
• Naive CD4+ T-cells +TGF-β, IL-2, CD3, CD28 stimulation: FOXP3
expression and supressive phenotype in vitro (proliferation) and in vivo
(prevent autoimmune disease in scurfy mice: 2bp insertion in the
FOXP3 gene, lymphoproliferative syndrome, lethal at the age of 3 to 5
weeks)
Human iTreg:
• TCR stimulation of naive human CD4+ FOXP3− T-cells in the presence
of TGFβ and IL-2 induces high levels of FOXP3 expression BUT human
iTregs fail to suppress responder CD4+FOXP3− T-cells. Restimulation:
IL-2, IFN-γ production
• Naive CD4+ T-cells from cord blood: similar results
• IL-10, vitamin D3, IDO, ATRA, epigenetic modifiers?
THE UNEXPECTED BIOLOGY OF IL-2
Dual roles of IL-2 in T-cell responses
Induction of immune response
Control of immune response
Prediction: What will be the consequence of eliminating IL-2 or the IL-2 receptor?
Surprising conclusion from IL-2 knock out mice:
the non-redundant function of IL-2 is in controlling immune responses (generalized autoimmune disease).
In humans, CD25 deficiency causes a syndrome described as IPEX-like. Extensive lymphocytic infiltration of
tissues, including lung, liver, gut and bone is observed, accompanied by tissue atrophy and inflammation. CD25deficient thymocytes fail to down-regulate levels of bcl-2, apoptosis in the thymus is markedly reduced,
resulting in expansion of autoreactive clones in multiple tissues.
FOXP3+ TREG CELLS THROUGHOUT LIFE
 The human thymus produces mature T-cells as early as the thirteenth week of gestation.
•
Most Tregs in cord blood have a naive phenotype, with high expression of CD45RA and low
expression of CD45RO.
•
The presence of a small number of effector Tregs (CD45RA-CD45RO+FOXP3hi) in cord
blood indicates activation in peripheral tissue.
•
Fetal Tregs have an early role in the maintenance of fetal self tolerance and in establishing
fetal–maternal tolerance.
 The proportion of naive Tregs among CD4+ T-cells declines with age:
•
Cord blood: 4-10%
•
Young adults: 1-4%
•
Elderly persons: 0.5-1.5%
 The prevalence of effector Tregs slightly increases with age:
•
Cord blood: 0-0.5%
•
Young adults: 1-2.5%
•
Elderly persons: 1-4%
 Because of thymic involution in adults, thymic output of T-cells is greatly reduced but still
detectable in elderly people, suggesting that the involuted thymic tissue may still be active.
MECHANISMS OF REGULATION
MOLECULES ASSOCIATED WITH TREG CELLS
MHCII/ANTIGEN
α-chain
β-chain
IL-2
CD80/86
CD25
LAG3
TCR complex
CD122
CD4
GITRL
IL-2R
CTLA-4
GITR
ATP
CD39
CD45RA/RO
FOXP3
CD73
AMP
IL-10
CD62L
TGF-β
GlyCAM-1
CD34
MadCAM-1
CCR,
CXCR
CCL
CXCL
CD127low/-CD49d-IL-2-
INHIBITORY CYTOKINES
TGF-β:
 Inhibits the proliferation and effector functions of T-cells.
 Suppresses the classical activation of macrophages, neutrophils and
endothelial cells.
 Stimulates production of IgA antibodies by inducing B-cells to switch to this
isotype. (IgA is the major antibody isotype required for mucosal immunity.)
 Promotes tissue repair after local inflammatory reactions (stimulate collagen
synthesis and angiogenesis).
 Membrane-tethered TGF-β can also mediate suppression by Treg cells in a
cell-cell contact-dependent manner.
 TGF-β knock out mice: progressive wasting syndrome and death 2 weeks
after birth.
INHIBITORY CYTOKINES
IL-10:
 Inhibits the production of IL-12 by activated dendritic cells and
macrophages and cell surface expression of co-stimulators and class II
MHC molecules.
 Inherited deficiencies of IL-10
(develops before 1 year of age).
or
IL-10
receptor:
severe
colitis
 Knockout mice lacking IL-10 either in all cells or only in regulatory T-cells also
develop colitis.
 IL-10 is especially important for controlling inflammatory reactions in
mucosal tissues, particularly in the gastrointestinal tract.
 The Epstein-Barr virus contains a gene homologous to human IL-10, and viral
IL-10 has the same activities as the natural cytokine (evolution of the virus,
survival advantage).
CYTOLYSIS
Treg-mediated target-cell killing was mediated by
granzyme A and perforin through the adhesion
molecule CD18.
doi:10.1038/nri2343
METABOLIC DISRUPTION
CYTOKINE DEPRIVATION
Tregs express all three components of the highaffinity IL-2R—CD25, CD122, and CD132—and IL-2 is
essential for Tregs homeostasis. Tregs may compete
with FOXP3− T-cells for IL-2, consume it, and inhibit
the proliferation of FOXP3−T-cells, resulting in a form
of apoptosis dependent on the pro-apoptotic factor
Bim.
doi:10.1016/j.immuni.2009.04.010
METABOLIC DISRUPTION
cAMP TRANSFER THROUGH GAP JUNCTION
Downregulation of miR-142-3p
which silences ADCY9 (adenylyl cyclase)
Treg
Tregs produce high
intracellular cAMP.
Downregulation of the Pde3b gene
(cAMP degrading phosphodiesterase 3b)
DOI: 10.1002/eji.201141578
cAMP upregulates CTLA-4.
Teff
cAMP facilitates the
expression of ICER
(inducible cAMP early
repressor).
ICER inhibits transcription of NFAT and
forms inhibitory complexes with preexisting
NFAT, thereby inhibiting NFAT-driven
transcription, including that of IL-2.
METABOLIC DISRUPTION
ADENOSINE NUCLEOSIDES
P2Y
Teff
cAMP
PRO-INFLAMMATORY
STIMULUS
P2X
Extracellular
Ectonucleoside triphosphate
ATP
diphosphohydrolase (E-NTPDase)
AMP
Adenosine 2A receptor
ADENOSINE
Ecto-5’-nucleotidase
TARGETING DENDRITIC CELLS
TARGETING DENDRITIC CELLS
LAG-3
Lymphocyte activation gene-3 (LAG-3, CD223) is a CD4-related
transmembrane protein that binds MHC II. LAG-3 engagement with
MHC class II inhibits DC activation inducing ERK-mediated
recruitment of SHP-1 that suppresses dendritic cell maturation and
immunostimulatory capacity. Because activated human T-cells can
express MHC class II, Treg-mediated ligation of LAG-3 on effectors
might also result in suppression.
doi:10.1016/j.immuni.2009.04.010
TARGETING DENDRITIC CELLS
CTLA-4
CTLA-4 promotes nuclear localization of Foxo3
transcription factor, which suppresses expression of
genes encoding IL-6 and TNF.
IDO (indoleamine-2,3-dioxygenase)
induction is also CTLA-4 dependent.
 IDO catalyses the degradation of the essential
amino acid L-triptophan to N-formylkynurenine, the
initial, rate-limiting step of tryptophan catabolism.
 Effector T-cells starved of tryptophan are unable to
proliferate
Blocking and go into G1 cell cycle arrest.
Downregulation
 Metabolites of tryptophan including kynurenine,
Transendocytosis
quinolinic acid, and picolinic acid are toxic to CD8+
and CD4+ Th1 cells.
TNF
TNF
CTLA-4 DEFICIENCY
 Deletion of CTLA-4 causes systemic autoimmunity in mice.
 CTLA-4 deficiency in Tregs alone is sufficient to cause fatal disease and maintenance
of its expression in activated effector T-cells is insufficient to prevent this
outcome.
 In humans, mutations of CTLA4 resulting in CTLA-4 haploinsufficiency cause a
complex immune dysregulation syndrome.
 Many patients previously diagnosed with CVID (Common variable immunodeficiency)
carry CTLA-4 mutation.
 CTLA-4 haploinsufficiency is characterized by infiltration of T-cells into the gut,
lungs, bone marrow, central nervous system, kidneys, and possibly other organs.
 Enteropathy, hypogammaglobulinemia, granulomatous lymphocytic interstitial lung
disease, respiratory infections, splenomegaly, thrombocytopenia, hemolytic anemia,
lymphadenopathy, psoriasis, thyroiditis, arthritis.
CTLA-4 HAPLOINSUFFICIENCY
10.1038/nm.3746
Tissue infiltration and lymphadenopathy in patients with CTLA4 mutations. (a,b) Duodenal biopsies from patient B.II.4 (a) and A.III.3 (b) stained for CD4. (c) Highresolution chest computed tomography scan of the lungs from patient E.II.3. Arrows point to granulomatous-lymphocytic infiltration in both lungs. (d) Pulmonary
lymphoid fibrotic lesions stained for CD4 in pulmonary biopsy from patient E.II.3. (e) Magnetic resonance imaging (MRI) of the pelvic area of patient A.III.3 with
two enlarged lymph nodes (arrows) measuring up to 4 cm. (f) Bone marrow biopsy from patient B.II.4 stained for CD4. (g) MRI of gadolinium-enhanced lesion
(arrows) in the cerebellum of patient A.III.1. (h) Resected cerebellar lesion from patient A.III.1 stained for CD3. Scale bars, 50 μm (a,b,d,f,h), 20 mm (c) and 50
mm (e).
CTLA-4 HAPLOINSUFFICIENCY
POTENTIAL THERAPY
SUMMARY
More than one mechanism
of Treg-mediated suppression
may operate for the control of a
particular immune response in a
synergistic or sequential manner
doi:10.1038/nri2343
REGULATORY T-CELL-BASED
IMMUNOTHERAPY
PHARMACOTHERAPIES
 Rapamycin: mTOR inhibitor, exploits the PI3 kinase pathway to preferentially expand Tregs.
Clinically, rapamycin increases the number of Tregs in lung and renal transplant patients.
 Anti-thymocyte globulin (ATG): T-cell-depleting polyclonal antibody, promotes Treg
generation in mice, supports allograft survival when combined with CTLA-4-Ig and rapamycin in
MHC-mismatched skin allograft model.
 Cyclosporine A and tacrolimus: Standard dosages of calcineurin inhibitors impair Tregs. Low
doses of calcineurin inhibitors: IL-2 production, may increase Treg numbers in the skin of
atopic dermatitis patients.
PHARMACOTHERAPIES
 IL-2/IL-2 monoclonal antibody complexes:
• 10-fold Treg expansion, resistance to experimental autoimmune encephalomyelitis and
islet allograft rejection in mice.
• Clininal trial (Phase 1): subcutaneous IL-2 to treat active chronic GVHD, daily low-dose
IL-2 was well-tolerated and led to sustained Treg expansion with improvement in GVHD
manifestations.
 Off-cell effect
• Pharmaceuticals that stimulate Tregs may also activate conventional T-cells.
• Phase I clinical trial of TGN1412 – a super-agonistic anti-CD28 antibody – caused
massive cytokine storm and multi-organ dysfunction.
CELLULAR THERAPY
The focus of intense research to treat autoimmune and graft-versus-host disease
Given the non-exclusivity of
CD25 and FOXP3 expression,
a number of other markers are in use
Depletion of CD127+ CD49d+ cells
CD25+CD45RA+FOXP3lo
(resting naive Treg)
Can be expanded and develop a
high frequency of functional
FOXP3+Tregs
Donor-derived APCs
CLINICAL APPLICATION OF TREG CELLS
Treg deficit associates with autoimmune disease development
 Failure to control islet-specific conventional T-cells results in type 1 diabetes mellitus (DM1).
 Risk of DM1 increases with the loss of FOXP3-expressing Tregs.
 Treg adoptive transfer to non-obese diabetic (NOD) mice can prevent the development of DM1.
 Clinical trial: DM1 children: autologous CD4+CD25highCD127−Treg infusion
• Decrease in the requirement of exogenous insulin in all the patients after 2 weeks.
• 4–5 months after Tregs administration: Of the 10 patients treated with Tregs, 8 were still
in clinical remission, with 2 patients out of insulin completely.
CLINICAL APPLICATION OF TREG CELLS
Transplantation
 Hematopoietic stem cell transplantation (HSCT)
•
Graft versus host disease
•
Inflammation often causes tissue
immunosuppressive pharmacotherapy.
damage
despite
routine
post-HSCT
• Ongoing clinical trials support the use of CD4+CD25+ Tregs to suppress GVHD.
 Solid organ transplantation
•
Graft rejection
• Tregs from mice prevented rejection of allogeneic skin grafts in nude mice given
CD25− T-cells.
•
The ONE Study: a multicenter Phase I/II clinical trial to evaluate the safety and
feasibility of various types of cell therapy including expanded Tregs in living-donor
kidney transplantation.
CLINICAL APPLICATION OF TREG CELLS
FOXP3+ Tregs impede the development of effective tumour
immunity
 A large number of CD4+CD25+FOXP3+ are present in tumours and draining lymph nodes
in patients with cancer.
 Decreased ratios of CD8+T-cells to CD4+CD25+FOXP3+ Tregs in tumours correlate with
poor prognosis.
 It has also been shown in numerous mouse models that depletion of Tregs enhances
antitumour immune responses, leading to the eradication of tumours.
 Studies in humans have shown that tumour antigen-specific CD4+T-cells can expand in
patients with cancer and healthy individuals following in vitro antigenic stimulation of
peripheral CD4+T-cells isolated from the individual after depletion of CD4+CD25+Tcells.
BARRIERS TO USE OF TREG CELLS FOR
IMMUNOTHERAPY
 Purity
•
Need to determine non-Treg contamination.
 Methylation status of the FOXP3 CNS2 region may indicate Treg purity and
stability.
 Potency
• As Tregs have many mechanisms of action, difficulty exists in elucidating which
mechanisms regulate a specific disease in an inflammatory environment
• In vitro assays – such as the ability of Tregs to inhibit conventional T-cell
proliferation – may inadequately describe the potency of cell preparations.
• CD4+ T-cells expanded in the presence of rapamycin were effective in vitro
(proliferation assay), but failed to function in vivo (xeno-GVHD model).
• Need to develop disease-specific Treg potency testing systems.
BARRIERS TO USE OF TREG CELLS FOR
IMMUNOTHERAPY
 Stability (after infusion)
•
FOXP3+ cells may lose FOXP3 expression.
•
“Ex-FOXP3” cells may display an activated conventional T-cell phenotype and become
pathogenic.
• IL-2 therapy might also promote Treg stability.
 Adverse effects
• Need to carefully examine the effect of Treg manipulation on infectious risk and
neoplasia.
• Interestingly investigators observed improved immunity to opportunistic pathogens in
trial of Treg infusion for GVHD prevention following HSCT.
• Numerous studies implicate Tregs in suppressing anti-tumor immunity.
THE END
PERIPHERAL TOLERANCE
Exposure of mature CD4+T-cells to an
antigen in the absence of co-stimulation or
innate immunity may make the cells
incapable of responding to that antigen.
Regulatory T lymphocytes are a subset of
CD4+ T-cells whose function is to suppress
immune responses and maintain selftolerance.
T-cells that recognize self antigens with
high affinity or are repeatedly stimulated by
antigens may die by apoptosis.
IDENTIFICATION OF THE TREG LINEAGE AND ITS CELL
SURFACE MARKERS BY DEPLETION EXPERIMENTS
Nude (athymic) mice (no T-cells)
+ transfer normal CD4+ T-cells
No autoimmunity
Nude (athymic) mice (no T-cells)
+ transfer CD5-depleted CD4+
T-cells (antibody + complement)
Autoimmunity
Treg
Teffector
Sakaguchi et al. showed in Ann rev immun. 2004
stomach, thyroid,
ovaries, testes
TREG DIFFERENTIATION
THYMIC REGULATORY T-CELLS
(tTreg)
Requirements for FOXP3 expression:
TCR signaling
High avidity self-specific TCR:
greater sensitivity to self-peptide–MHC than
potentially pathogenic autoreactive T-cells.
Co-stimulation
Decreases in frequencies of Tregs in
CD28-deficient and CD80-CD86-deficient
mice.
Cytokine-mediated signals
IL-2, IL-7, IL-15
Control of systemic and
tissue-specific autoimmunity
Ongoing TCR
engagement
TREG DIFFERENTIATION
Local immune supression
Mucosal, oral, fetal tolerance
PERIPHERAL REGULATORY
T-CELLS (pTreg)
Low level TCR signaling:
Suboptimal DC activation
Sub-immunogenic doses of agonist peptide
Cytokine-mediated signals
IL-2, TGF-β
While CD28 signaling is critical for central
selection of Tregs, peripheral Treg induction is,
in contrast, inhibited by strong CD28 ligation.
ATRA
Synergizes with TGF-β
Sufficient to allow pTreg development even
in the presence of high levels of costimulation.
tissue-specific
or foreign
antigen
FOXP3+pTreg
FOXP3+pTreg
EPIGENETIC CONTROL OF FOXP3 EXPRESSION
Partial demethylation
p
doi: 10.3389/fimmu.2013.00169
Classic TATA and
CAAT-box-containing promoter
CNS1 (Conserved non-coding sequence):
TGF-β sensor
CNS1 deficient mice:
No detectable tissue-specific autoimmunity BUT
impaired pTreg generation
Th2-type pathologies at mucosal sites
Increased embryo resorption
CNS2: TREG-SPECIFIC DEMETHYLATED REGION (TSDR)
MAINTENANCE OF STABIL FOXP3 EXPRESSION.
CNS2-deficient mice: FOXP3 induction in the thymus and in the periphery
was unimpaired BUT progressive lost of FOXP3 expression upon division.
FOXP3 is recruited to CNS2
CNS3: NF-κB family members bind to this element and
facilitate the activity of the FOXP3 promoter.
CNS3 deficient mice remain healthy despite decrease
in repertoire of Treg cells.
METABOLIC DISRUPTION
cAMP TRANSFER THROUGH GAP JUNCTION
Downregulation of miR-142-3p
which silences ADCY9 (adenylyl cyclase)
Treg
Tregs produce high
intracellular cAMP.
Downregulation of the Pde3b
(cAMP degrading phosphodiesterase 3b)
gene
DOI: 10.1002/eji.201141578
cAMP upregulates CTLA-4.
Teff
cAMP facilitates the
expression of ICER
(inducible cAMP early
repressor)
ICER inhibits transcription of NFAT and forms
inhibitory complexes with preexisting
NFAT, thereby inhibiting NFAT-driven
transcription, including that of IL-2.
TARGETING DENDRITIC CELLS
CTLA-4
In addition to direct or indirect modulation of CD80
and CD86 expression, these signals can also promote
nuclear localization of Foxo3 transcription factor,
which suppress expression of genes encoding IL-6 and
TNF.
IDO (indoleamine-2,3-dioxygenase)
induction is also CTLA-4 dependent.
 IDO catalyses the degradation of the essential
amino acid L-triptophan to N-formylkynurenine, the
initial, rate-limiting step of tryptophan catabolism.
 Effector T-cells starved of tryptophan are unable to
proliferate and go into G1 cell cycle arrest.
 Metabolites of tryptophan including kynurenine,
quinolinic acid, and picolinic acid are toxic to CD8+
and CD4+ Th1 cells.
Blocking
Downregulation
Transendocytosis