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IMMUNOLOGICAL TOLERANCE
ARPAD LANYI PhD
[email protected]
The response of the immune system
to the stimuli of the outer and inner environment
Environment
Immune system
Tolerance
Self
Non-self
Dangerous
Pathogenic
Immune
response
Self/non-self discrimination is a central issue in immunology
Immunological tolerance
Definition:
Lack of adaptive immune response against a given antigen.
The interaction of the antigen with the lymphocytes induces unresponsiveness.
ANTIGEN SPECIFIC!!!
Unlike immunosuppression
Why is this important?
-All individuals are tolerant to their own antigens (self tolerance)
-Failure of self tolerance results in autoimmunity
-Terapeutic potential:
Treat autoimmune diseases, allergic reaction or even tissue rejection
Immunological tolerance
CENTRAL TOLERANCE:
Development of immunological tolerance begins in the primary
(central) lymphoid organs (bone marrow and thymus) as an integral
part of normal lymphocyte development
PERIPHERAL TOLERANCE:
Elimination/inhibition of autoreactive
tolerance
clones
escaping
central
T-CELL TOLERANCE
CENTRAL TOLERANCE
OWING TO AIRE CENTRAL T-CELL TOLERANCE IS HIGHLY
EFFECTIVE
PERIPHERAL TOLERANCE
Autoreactive T-cells encountering with self antigen in
the peripheral tissues may be inactivated, deleted, or
suppressed by regulatory T-cells
In the absence of infection
dendritic cells presenting self antigens
do NOT express co-stimulatory molecules at high levels
Antigen recognition in the absence of
co-stimulation leads to a state of T-cell ANERGY
ANERGY:
FUNCTIONAL UNRESPONSIVENESS
Phosphatases
Ubiquitin ligases
Binding of TCR to self antigens with high affinity
in the absence of co-stimulation
may lead to BIM-mediated apoptosis
REPEATED stimulation of T-cells results in
FAS-meditated apoptosis
APOPTOSIS
Bimmediated
apoptosis
Activationinduced cell
death
REGULATORY T LYMPHOCYTES
EARLY EXPERIMENTAL MODELS SUGGESTING THE
PRESENCE OF REGULATORY T-CELLS
Murine neonatal thymectomy at day 3 leads to destruction of ovary
(autoimmune)
This phenotype can be prevented by injection of CD4+ T-cells/thymus
transplantation
Thymectomy did not cause autoimmunity if performed on day 1 or day 7
after birth
Shortly after birth autoreactive T-cells leave the thymus, followed by
suppressor T-cells a few days later, which can inhibit the activity of the
autoreactive clones
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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
X
INHIBITION OF AUTOIMMUNE
PROCESSES BY CD25+CD4+TR CELLS
X
FOXP3: MORE THAN A LINEAGE MARKER
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)
The function of
CD4+FOXP3+CTLA-4+CD25+ regulatory T-cells
is to suppress immune responses and
maintain self-tolerance
DIFFERENTIATION
OF REGULATORY T-CELLS
DIFFERENTIATION OF
THYMUS-DERIVED
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
• Strong co-stimulation
• IL-2
Foxp3 induces the expression of
CTLA-4 and CD25 and inhibits the
expression of IL-2
Hassall’s corpuscles instruct dendritic cells to induce
the development of regulatory T-cells in human
thymus
Watanabe et al. Nature 436, 1181
doi:10.1038/nature03886
DIFFERENTIATION OF
PERIPHERAL REGULATORY TCELLS (pTreg)
pTreg
STAT5
FOXP3
Suboptimal TCR-activation
Low level co-stimulation
TGF-β, IL-2
Naive CD4
T-cell
IL-12
IFN-γ
IL-4
Th1
STAT1/4
T-bet
Th2
STAT6
GATA-3
IL-6, IL-12,
IL-23, TGF-β
IL-1
IL-6
TGF-β
Th17
STAT3
RORγT
Tfh
Bcl6
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
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)
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.
X
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
SUMMARY: Treg DIFFERENTIATION
tTreg
pTreg
High affinity TCR
specific for self antigen
Self or non-self
antigen-specific TCR
Co-stimulation
Suboptimal TCR signal
IL-2
Low level co-stimulation
Control of systemic and
tissue-specific
autoimmunity
IL-2, TGF-β
Control of local tissuespecific autoimmunity
MECHANISMS OF REGULATION
MECHANISMS OF IMMUNOLOGICAL
TOLERANCE MAINTAINED BY
REGULATORY T CELLS
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 cellcell 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
CTLA-4
COMPETITION
TRANS-ENDOCYTOSIS
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 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.
TNF
TNF
doi:10.1038/ni.1818
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
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CTLA-4 HAPLOINSUFFICIENCY
Duodenum
MRI pelvic
Bone marrow biopsy
Lung
MRI Cerebellum
Cerebellum
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
Schubert etal. Nat. Med 2014
doi:10.1038/nm.3746
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CTLA-4 HAPLOINSUFFICIENCY
POTENTIAL THERAPY
SUMMARY: PERIPHERAL MECHANISMS
OF T-CELL TOLERANCE
B-CELL TOLERANCE
CENTRAL TOLERANCE
NEGATIVE SELECTION
~75% of immature B-cells have some
affinity for self antigens
Many tissue-specific antigens are
not expressed in the bone marrow
30-40% of transitional B-cells
leaving the bone marrow are still
autoreactive
PERIPHERAL MECHANISMS OF
B-CELL TOLERANCE
SELECTION OF TRANSITIONAL B-CELLS
doi:10.3390/antib3010116
ESTABLISHING T-CELL TOLERANCE IS ESSENTIAL TO
MAINTAIN PERIPHERAL B-CELL TOLERANCE
AUTOANTIBODY PRODUCTION IS DEPENDENT ON THE
AVAILABILITY OF AUTOREACTIVE T-CELLS
In the absence of T-cell help autoreactive
B-cells are retained in the T-cell zone and
die by apoptosis
LACK OF DANGER SIGNALS
RECEPTOR BLOCKADE: HIGH ZONE TOLERANCE

THANK YOU
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
X
INHIBITION OF AUTOIMMUNE
PROCESSES BY CD25+CD4+TR CELLS
X
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.
REGULATORY T-CELL-BASED
IMMUNOTHERAPY
X
LOSS OF REGULATION OF AUTOREACTIVE T-CELLS
RESULTS IN AUTOIMMUNITY
)
doi:10.1038/nri2889
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
 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
Donor APC
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
•
Phase I/II clinical trials 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.