Mechanisms of tolerance induction - BNITM · . central and peripheral mechanisms of tolerance induction ... into nude mouse

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




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


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


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 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




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




peripheral mechanisms of tolerance



•  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




•  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


•  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


•  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!

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Mechanisms of tolerance induction - BNITM · . central and peripheral mechanisms of tolerance induction ... into nude mouse