Post on 28-Jun-2018
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...
• how does the immune system „decide“ against what to react?
T
self antigens
foreign pathogen
reactivity
tolerance
harmless foreign antigen
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.
• Ehrlich P & Morgenstern J: Über Haemolysine. Fünfte Mittheilung. Berliner Klinische Wochenschrift 38: 251-257 (1901)
www.uab.edu/reynolds/IMAGES/Ehrlich.jpg
central and peripheral mechanisms of tolerance induction
central tolerance deletion of autoreactive lymphocytes in the central lymphatic organs
APS-1 syndrome
IPEX syndrome
peripheral tolerance additional mechanisms in the periphery
B
• 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
from: Kyewski B, Klein L. Ann Rev Immunol. 2006
10-100 cells/d
20 cell divisions 5*10E7 cells/d
1*10E6 cells/d
induction of central tolerance in T cells
aus: Palmer E: , Negative Selection - Clearing out the Bad Apples from the T-cell Repertoire Nature Rev Imm 3 (2003)
> 90%
5% 2-5%
too weak interaction with pMHC USELESS !
too strong interaction with pMHC HARMFUL !
intermediate reactivity USEFUL !
failing positive selection
CD4+/CD8+ double-positive
CD4+ or CD8+ single-positive
induction of central tolerance in T cells
aus: Palmer E: , Negative Selection - Clearing out the Bad Apples from the T-cell Repertoire Nature Rev Imm 3 (2003)
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
induction of central tolerance in T cells
modified from: Colin R.A. Hewitt, University of Leicester, UK
probability of death
affinity of the TcR/pMHC interaction low high
Useless Useful Harmful
Death by Neglect
positive selection
negative selection
• 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
Ishii, T. et al. J Clin Endocrinol Metab (2000)
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.
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 AIRE-dependent 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
aus: Anderson MS et al. Science (2002)
Aire-ko thymus transplanted into nude mouse
Expression of Aire in the thymus is critical for prevention of autoimmunity
wild-type thymus transplanted into nude mouse
• 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)
CD4
CD25
A population of CD4+/CD25+ T cells has a dominant suppressive function in vitro
• 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)
aus: Hori S et al. Science 2003 number of added cells (x 104)
Prol
ifera
tion
mouse lymph node cells
CD4+/25+ nTregs have a suppressive function in vivo
• 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)
naive T-Zellen
naive T cells
+ CD25+ T cells
CD4+/25+ nTregs have a suppressive function in vivo
• 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)
naive T-Zellen
naive T cells
+ CD25+ T cells
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
aus: Nieves DS et al. Arch Dermatol. 2004
lymphocytic infiltrate of the skin of a patient with IPEX syndrome
the transcription factor FOXP3 is the genetic defect of the IPEX syndrome
The scurfy mouse: a natural animal model of the IPEX syndrome
aus: Smyk-Pearson SK et al. Clin Exp Immunol 2003
• 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
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)
• 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
aus: Wan Y. PNAS 2005
from: Sakaguchi S et al. Nature Rev. Immunol. 2009
FoxP3+ Tregs in CD4+ cells
DG_V23
0 102 103 104 105
<APC-A>: FoxP3
0
102
103
104
105
<Per
CP
-Cy5
-5-A
>: C
D4
11.486.2
0 102 103 104 105
<Pacific Blue-A>: CD3
0
102
103
104
105
<Per
CP
-Cy5
-5-A
>: C
D4
41.2
CD3 CD
4 FoxP3 CD
4
FoxP3+ Tregs are CD25hi and CD127lo
0 102 103 104 105
<APC-A>: FoxP3
0
102
103
104
105
<PE-
A>: C
D25
0 102 103 104 105
<APC-A>: FoxP3
0
102
103
104
105
<FIT
C-A
>: C
D12
7
DG_V23
FoxP3 CD
25
FoxP3
CD
127
0 102 103 104 105
<PE-A>: CD25
0
102
103
104
105
<FIT
C-A
>: C
D12
7
76.1 1.93
9.4812.5
CD25 CD
127
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
0 102 103 104 105
<PE-A>: CD25
0
102
103
104
105
<PE-
Cy7
-A>:
CD
39
7.4 0.36
2.0290.2
0 102 103 104 105
<PE-A>: CD25
0
102
103
104
105
<PE-
Cy7
-A>:
CD
39
10.4 41.5
38.49.75
FoxP3- Zellen FoxP3+ Zellen
CD25 CD
39
CD25 CD
39
Positive role of P2X7 and CD39 on Treg suppressive function ?
CD73
ADO CD39
ADP AMP
A2a
(-)
Activated effector T cells
Activation
CD4 regulatory T cells
Foxp3
P2X7
CD25
ATP
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
Treg
autoimmunity infection
allergy transplantation tolerance
immunity to
tumors
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
• 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)
Tumor-inflitrating lymphocytes
different populations of regulatory T cells
Tnaive
T Thymus-
zelle
Thymus
APC “natürliche”
Treg
induzierte Treg
TGFβ
Treg
Treg
TGFβ, IL-10
TGFβ, IL-10
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.
Institute of Immunology, Hamburg
- Fritz Koch-Nolte - Hans-Willi Mittrücker - Eva Tolosa
- Bernhard Fleischer
INSERM U905, Rouen
- Michel Seman - Sahil Adriouch - Sandra Hubert
thank you!