Mechanisms of tolerance induction
Friedrich Haag
Institute of Immunology, University Medical Center Hamburg-Eppendorf
2nd South-Eastern European Immunnology School, Dubrovnik, Croatia, 2010
The immunologic dilemma
•  cells of the immune system
are surrounded by a sea of
„self“, in which a few tiny
islands of „foreign“ are
swimming...
self antigens
tolerance
T
reactivity
harmless foreign
antigen
•  how does the immune
system „decide“ against what
to react?
foreign pathogen
the conceptual history of tolerance and autoimmunity
•  The organism disposes of certain
facilities that prevent that the
immune reaction, which is elicited
so easily by the most different cell
types, be directed against its own
elements, and that autotoxins be
produced...
•  ...so that it is justified to speak of a
horror autotoxicus of the human
organism.
• 
www.uab.edu/reynolds/IMAGES/Ehrlich.jpg
Ehrlich P & Morgenstern J: Über Haemolysine.
Fünfte Mittheilung. Berliner Klinische
Wochenschrift 38: 251-257 (1901)
central and peripheral mechanisms of tolerance induction
central tolerance
deletion of autoreactive
lymphocytes in the
central lymphatic organs
APS-1 syndrome
B
IPEX syndrome
peripheral tolerance
additional mechanisms in the
periphery
•  Central tolerance
–  induction of central tolerance in T cells
•  central tolerance to tissue-restricted antigens
–  induction of central tolerance in B cells
•  Peripheral tolerance
–  „recessive“ tolerance
–  „dominant“ tolerance
•  Central tolerance
–  induction of central tolerance in T cells
•  central tolerance to tissue-restricted antigens
–  induction of central tolerance in B cells
•  Peripheral tolerance
–  „recessive“ tolerance
–  „dominant“ tolerance
maturation and selection of T lymphocytes in the thymus
20 cell divisions
5*10E7
cells/d
10-100
cells/d
1*10E6
cells/d
from: Kyewski B, Klein L. Ann Rev Immunol. 2006
induction of central tolerance in T cells
failing
positive selection
> 90%
CD4+/CD8+
double-positive
too weak interaction
with pMHC
USELESS !
CD4+ or CD8+
single-positive
5%
2-5%
too strong interaction
with pMHC
HARMFUL !
intermediate reactivity
USEFUL !
aus: Palmer E: , Negative Selection - Clearing out the Bad Apples from the T-cell Repertoire Nature Rev Imm 3 (2003)
induction of central tolerance in T cells
positive selection
-  cortex (cortical epithelial cells)
- reactivity of the TCR with self-MHC molecules
-  survival signal
negative selection
- mostly medulla
- DCs and medullary epithelial cells (mTECs)
- strong reactivity of the TCR with MHC + self
-antigen
- death signal
aus: Palmer E: , Negative Selection - Clearing out the Bad Apples from the T-cell Repertoire Nature Rev Imm 3 (2003)
induction of central tolerance in T cells
Useless
Death by
Neglect
Useful
Harmful
positive
selection
probability of
death
negative
selection
high
low
affinity of the TcR/pMHC interaction
modified from: Colin R.A. Hewitt, University of Leicester, UK
•  Central tolerance
–  induction of central tolerance in T cells
•  central tolerance to tissue-restricted antigens
–  induction of central tolerance in B cells
•  Peripheral tolerance
–  „recessive“ tolerance
–  „dominant“ tolerance
central tolerance to tissue-restricted antigens
APS-1 or APECED syndrome
what about tolerance to tissue-restricted antigens
not expressed in the thymus ?
The APS-1 syndrome: the importance of central mechanisms for
maintenance of tolerance against tissue restricted antigens
•  autoimmune polyglandular syndrome I
–  also designated as APECED: (autoimmune
polyendocrinopathy-candidiasis-ectodermal
dystrophy)
•  autosomal recessive inheritance
•  classical triad of symptoms:
– 
– 
– 
– 
adrenal insufficiency (Addison‘s disease)
hypoparathyroidism
generalised candidiasis of mucous membranes
further endocrine oirgans may be involved
•  usually begins in childhood
–  variable penetrance of symptoms
Figure 1. Chronic mucocutaneous candidiasis in two
siblings. Thrush (A) and ungual candidiasis (B) in case 1
at 11 yr of age. Ungual candidiasis (C) in case 2 at 9 yr
of age.
Ishii, T. et al. J Clin Endocrinol Metab (2000)
The AIRE (autoimmune regulator) gene : cause of the APS-1
syndrome
• 
• 
transcription factor
expressed mainly by medullary
thymic epithelial cells (mTECs)
aus: Peterson P et al. Clin Exp Immunol (2004)
The AIRE (autoimmune regulator) gene : cause of the APS-1
syndrome
aus: Anderson MS et al. Science (2002)
Mathis D, Nat Rev. Immunol. 2007
expression of tissue-restricted antigens in the thymus
• 
Aire induces the ectopic expression
of tissue-restricted antigens (TRAs)
in medullary thymic epithelial cells
(mTECs)
• 
presumably hundreds of TRAs are
expressed in mTECs in an AIREdependent manner
• 
causes negative selection of
autoreactive T cells
• 
mouse model: targeted deletion of
the Aire gene leads to autoantibodies
and lymphocytic infiltrations in
endocrine glands
Expression of a liver-specific antigen by two mTECs (in
situ hybridisation)
Abbildung: L. Klein, Institute of Molecular Pathology, Wien
mechanisms of AIRE-driven presentation
•  negative selection of T cells
specific for TRAs may
occur by two mechanisms
–  direct presentation of AIRE
-driven TRAs by mTECs
–  uptake of apoptotic mTECs
by medullary DCs, and
cross-presentation
mouse model for Aire deficiency
Aire-ko thymus transplanted
into nude mouse
wild-type thymus transplanted
into nude mouse
Expression of Aire in the
thymus is critical for
prevention of
autoimmunity
aus: Anderson MS et al. Science (2002)
•  Central tolerance
–  induction of central tolerance in T cells
•  central tolerance to tissue-restricted antigens
–  induction of central tolerance in B cells
•  Peripheral tolerance
–  „recessive“ tolerance
–  „dominant“ tolerance
Induction of central tolerance in B cells
selection at the pre-BCR (checkpoint 1)
•  pre-BCR checkpoint mediates
–  positive selection
•  successful H-chain expression
–  presumably also negative selection
•  large part of the poly- and
autoreactive repertoire is lost at
this stage
Summary: Central tolerance
•  Central tolerance mechanisms regulate self-reactivity in both T and B
lymphocytes
•  Central control of self-reactivity is more stringent for T than for B cells
•  the combination of positive and negative selection in the thymus
selects for TCRs with intermediate affinity to self-pMHC
•  the autoimmune regulator AIRE drives ectopic expression of many
tissue-restricted antigens by mTECs, thus allowing for negative
selection against TCRs that could react with these antigens
peripheral mechanisms of tolerance
•  Central tolerance
–  induction of central tolerance in T cells
•  central tolerance to tissue-restricted antigens
–  induction of central tolerance in B cells
•  Peripheral tolerance
–  „recessive“ tolerance
–  „dominant“ tolerance
peripheral mechanisms of tolerance
•  Central tolerance
–  induction of central tolerance in T cells
•  central tolerance to tissue-restricted antigens
–  induction of central tolerance in B cells
•  Peripheral tolerance
–  „recessive“ tolerance
•  „ignorance“
•  anergy
–  „dominant“ tolerance
peripheral tolerance: recessive mechanisms
•  „Ignorance“
–  the self-reactive lymphocyte is present in the periphery, but does
not „see“ the antigen it is directed against
–  immune-privileged sites
•  brain
•  eyes
•  testes
•  placenta and fetus
–  control of T-cell trafficking to tissues
•  naive cells recirculate through secondary lymphatic organs and
bloodstream, but do not enter into tissues under normal
conditions
peripheral tolerance: recessive mechanisms
•  anergy
–  full activation of T cells requires
costimulation through CD28 in
addition to TCR ligation
–  TCR ligation in the absence of
costimulation leads to inability to
express effector functions like
cytokine secretion, and makes
the cell unresponsive to further
stimulation
–  control of the expression of the
costimulatory molecules CD80
and CD86 (B7) is a major
mechanism of peripheral
tolerance
Miller et al. Nature Rev. Immunol. 2007
danger signals generate immunogenic DCs via TLR signals
•  immunogenic phenotype of DCs
–  pathogens, necrotic cells
–  „danger signals“, sensed by TLRs
apoptotic cells generate tolerogenic DCs
•  tolerogenic phenotype of DCs
–  apoptotic cells
–  inhibits TLR signalling
•  Central tolerance
–  induction of central tolerance in T cells
•  central tolerance to tissue-restricted antigens
–  induction of central tolerance in B cells
•  Peripheral tolerance
–  „recessive“ tolerance
–  „dominant“ tolerance
•  identification of cell populations with suppressive
(„regulatory“) features
•  most prominent example: CD4+/CD25+ natural
regulatory T cells (nTregs)
A population of CD4+/CD25+ T cells has a
dominant suppressive function in vitro
CD25
mouse lymph node
cells
Proliferation
CD4
• 
stimulation of naïve T cells over several days
with anti-CD3 antibodies induces
proliferation
• 
co-cultivation of anti-CD3 stimulated naïve T
cells with
–  CD25- cells (green population)
–  CD25+ cells (blue population)
number of added cells (x 104)
aus: Hori S et al. Science 2003
CD4+/25+ nTregs have a suppressive function in vivo
naive
T cells
+
CD25+
T cells
naive
T-Zellen
• 
adoptive transfer of naïve T cells into an immunodeficient host (lacking T cells of its own)
causes autoimmune colitis in the host (blue and red lines)
• 
colitis is prevented by co-transfer of purified CD4+/CD25+ regulatory T cells (green line)
aus: Powrie F. Immunol Rev (2005)
CD4+/25+ nTregs have a suppressive function in vivo
naive
T cells
+
CD25+
T cells
naive
T-Zellen
• 
normal mice contain sufficient autoreactive peripheral T cells to induce autoimmune
disease in a host, if they are not restrained
• 
restraint of autoreactive autoimmunity is performed by the population of regulatory T cells
aus: Powrie F. Immunol Rev (2005)
The IPEX syndrome
• 
• 
• 
• 
immune dysfunction
polydendocrinopathy
enteropathy
X-linked
aus: Nieves DS et al. Arch Dermatol. 2004
The IPEX syndrome
•  usually lethal within the first few years of life
•  multiple autoimmune phenomena, mainly affecting
endocrine glands
lymphocytic infiltrate of the skin of a
patient with IPEX syndrome
aus: Nieves DS et al. Arch Dermatol. 2004
the transcription factor FOXP3 is the genetic defect
of the IPEX syndrome
The scurfy mouse: a natural animal model of the IPEX syndrome
•  the scurfy mouse has multiple
autoimmune phenomena
reminiscent of the IPEX
syndrome
•  the gene defect of the scurfy
mouse was mapped to the
Foxp3 gene
•  deletion of this gene causes the
same phenotype
•  genetische Komplementierung
mit einem intakten Foxp3-Gen
führte zum Verschwinden der
Symptome
aus: Smyk-Pearson SK et al. Clin Exp Immunol 2003
FOXP3 controls the development of a population
of „regulatory“ T cells
•  Foxp3+ cells are
characterized by high
expression of the alpha
chain of the IL-2 receptor
(CD25)
aus: Wan Y.
PNAS 2005
•  this population is missing in
patients with IPEX
syndrome
•  this population is designated
as CD4+/CD25+/FoxP3+
regulatory T cells (Tregs)
aus: Torgerson TR. Springer Sem Immunol 2006
from: Sakaguchi S et al. Nature Rev. Immunol. 2009
FoxP3+ Tregs in CD4+ cells
105
41.2
<PerCP-Cy5-5-A>: CD4
<PerCP-Cy5-5-A>: CD4
105
104
103
102
104
103
102
0
0
102
103
104
<Pacific Blue-A>: CD3
105
CD4
CD4
0
CD3
11.4
86.2
0
FoxP3
102
103
104
<APC-A>: FoxP3
DG_V23
105
105
104
104
104
3
10
<FITC-A>: CD127
105
<FITC-A>: CD127
105
3
10
102
0
0
0
FoxP3
102
103
104
<APC-A>: FoxP3
105
0
102
FoxP3
103
104
<APC-A>: FoxP3
105
CD127
102
0
76.1
1.93
12.5
9.48
103
102
CD127
CD25
<PE-A>: CD25
FoxP3+ Tregs are CD25hi and CD127lo
0
CD25
102
103
104
<PE-A>: CD25
DG_V23
105
how do Tregs exert their suppressive effects ?
•  many possible mechanisms:
–  production of suppressive cytokines (e.g. IL-10, TGFβ)
–  sequestration of IL-2 from effector cells
•  high expression of CD25
–  production of adenosine from extracellular ATP
•  via CD39 (ATPase) and CD73 (nucleotidase)
•  adenosine acts on suppressive A2A receptors present on
activated T cells
–  expression of suppressive cell surface markers (e.g. CTLA4)
•  in vitro evidence for all of these mechanisms
•  all may be important in distinct situations
CD39 differenziert Subpopulationen in FoxP3+ Zellen
FoxP3- Zellen
7.4
0.36
<PE-Cy7-A>: CD39
104
103
0
0
0
2.02
102
103
104
<PE-A>: CD25
105
41.5
9.75
38.4
103
102
CD25
10.4
104
102
90.2
CD39
105
CD39
<PE-Cy7-A>: CD39
105
FoxP3+ Zellen
CD25
0
102
103
104
<PE-A>: CD25
105
Positive role of P2X7 and CD39 on Treg suppressive function ?
Activation
P2X7
ATP
A2a
ADP
AMP
CD25
(-)
CD39
Foxp3
ADO
CD73
CD4 regulatory T cells
Activated effector T cells
Michel Seman, Rouen
Patienten mit MS weisen erniedrigte Zahlen von CD25+/CD39+ T
-Zellen auf
Borsellino G et al, Blood 2007
how do Tregs exert their suppressive effects ?
•  one important mechanism is the negative regulation of dendritic cell
maturation
• 
in the absence of Tregs, effector T
cells may exert an „adjuvant
effect“ on DCs, causing their
maturation and facilitating the
activation of autoreactive T cells
• 
this is blocked by Tregs, keeping
DCs in an immature (tolerogenic)
state
Wing. Nature Rev. Immunol 2010
how do Tregs exert their suppressive effects ?
•  The expression of CTLA-4
may be a „core“
mechanism of Treg
function
•  selective deletion of CTLA-4
expression in Tregs
induces a similar
phenotype as total deletion
of Tregs
•  Expression of CTLA-4 by
Tregs may be a master
mechanism to suppress
maturation of DCs
Wing. Nature Rev. Immunol 2010
autoimmunity
allergy
infection
Treg
immunity
to
tumors
transplantation
tolerance
in vivo evidence for role of Tregs in suppressing immunity to
tumors
•  depletion of Tregs
leads to retardation of
tumor growth (A)
Hubert S et al. J Exp Med (in press)
in vivo evidence for role of Tregs in suppressing immunity to
tumors
Tumor-inflitrating lymphocytes
•  depletion of Tregs
leads to retardation of
tumor growth (A)
•  and to increased
expression of
cytotoxic effector
molecules (granzyme
B) by tumor
-infiltrating
lymphocytes (C).
Hubert S et al. J Exp Med (in press)
different populations of regulatory T cells
Thymus
Thymuszelle
T
APC
“natürliche”
Treg
Treg
Tnaive
TGFβ
TGFβ,
IL-10
Treg
TGFβ,
IL-10
induzierte
Treg
Summary: peripheral tolerance
•  central tolerance is important, but leaky:
–  self-reactive T and B cells exist in the periphery of normal healthy subjects
and need to be kept in check by peripheral tolerance mechanisms
•  peripheral tolerance mechanisms can be divided into recessive (cell
-inherent) and dominant (suppression of one cell by another)
mechanisms
•  CD4+/CD25+ natural Tregs are the paradigm of dominant peripheral
tolerance. Deletion of these cells results in multiple and severe
autoimmune phenomena
•  nTregs develop in the thymus and are controlled by the master
transcription factor FoxP3.
•  among many other possible mechanisms of action, control of DC
maturation by CTLA-4 expressed on Tregs may be a master
mechanism by which nTregs exert their suppressive effects.
thank you!
Institute of Immunology,
Hamburg
INSERM U905,
Rouen
-  Fritz Koch-Nolte
-  Hans-Willi Mittrücker
-  Eva Tolosa
-  Michel Seman
-  Sahil Adriouch
-  Sandra Hubert
-  Bernhard Fleischer