Tolerance: an intrinsic property of the immune system.

Tolerance is the failure to respond against an

antigen

Self-tolerance keeps host responses under

constraint. It controls immune cells and

molecules to protect host tissues.

The dilemmas of lymphocytes: to attack or not to attack

Layers of tolerance

Central tolerance

Peripheral anergy

Peripheral clonal deletion

Cytokine deviation

Immune-privileged sites

Ignorance

Dominant toleranceRegulatory T cells (natural occurring, adaptive)

IDO-expressing DCs

Regulation by anti-inflammatory peptides

??

However, many –if not all- of these mechanisms

can fail in a significant proportion (>5%) of

humans

Subtle changes in cytokine profile can shift the balance between controlled response and

autoimmunity

Cytokine effect depends on local milieu and target immune and tissue cells

In a sense all healthy tissues can be concidered immune privileged !

Physical barriers to exclude naive lymphocytes-lack of molecules that drive

lymphoc. migration

Production of anti-infammatory factors (TGFβ)

Expression of death ligands (FasL) to kill activated T lymphocytes

...but even these can fail (eg multiple sclerosis, sympathetic opthalmia)

Tolerance in immune-privileged sites

A rough outline of lymphocyte tolerance

Thymus serves for establishing central T cell tolerance and production of regulatory T cells (Tregs)

Numberof cells

Affinity of TcR/MHC interactionLow High

UselessNeglect

UsefulPositively select

HarmfulNegatively select

Negative selection: survival of the fittest

Y O U A R E H E R E

But if developing T cells “see” self-Ags in the thymus, what about tissue-specific antigens?

AIRE: AutoImmune REgulatorAPECED (Autoim. PolyEndocrino-Pathy Candidiasis Ectodermal Dystrophy)

Ocasionally potentially autoreactive naive T cells escape in the periphery

PeripheryThymus

mTEC DC

release

parenchymal cell

effector cells

uptake

T

T

T

T

activation

T TTTT

T TT

TCR- signaling alone on naive T cells promotes tolerance rather than immune response

(a safe-guard mechanism to prevent aberrant activation)

2nd signal is enhanced and 3rd signal is provided by

inflammation/TLR activation leading to DCs

maturation

Investigation of T cell anergy

mechanisms was based originally on two

main models

1. Activation of Th cell lines only through

TCR.

Anergy reversible by IL-2.

Stimulus withdrawal does not reverse

anergy.

AgTCR-tg T cells

2. Adaptive tolerance

Anergy irreversible by IL-2

Ag withdrawal reverses anergic phenotype

Similar anergic phenotypes in TCR x self-Ag tg models

#

T i m e

Anergic T cells.

Anergic phenotypes are

heterogeneous and can be

partial (split tolerance)

Relevance of these to naturally occurring T cell tolerance?

Other situations that drive towards peripheral T cell tolerance in vivo

High doses of antigen (sterile) (actually in the absence of adjuvants, eg

TLR-ligands)

Altered peptide ligands

Repeated doses of antigen (sterile)

Persistent infections (HIV, CMV, hepatitis etc) (exhaustion)

Chronic exposure to Ag

Route of administration (oral (strong), intravenous )

Tumor antigens/tumor evasion of immune response

Strange enough lack of co-stimulatory molecules such as CD28 or TNFR2

impairs T cell tolerance development!!

F5

antigen

F5/NP

F5 TCR-tg mice. The majority of T cells are CD8 and express the F5 T cell receptor (F5 TCR) which recognizes the NP68 peptide (on MHC-I)

NP-transgenic mice . Endogenous expression of NP protein in amny tissues/cell types (part of it is the NP68 nonaner peptide)

F5/NP mice (double-transgenic). A model for CD8 T cell responses to self antigens

An example of TCR-self Ag double tg mouse as a model for studying T cell tolerance

5.4%26.1%

F5 F5/NPAMFI:544

85.7%

MFI:120

37.9%

MFI:454

34.8%

MFI:91

16.2%

F5/Rag1-/- F5/NP/Rag1-/-

CD44

CD69

Reduced numbers of F5 T cells in F5/NP mice (peripheral clonal deletion)

Vβ11

CD8

0

20

40

60

80

100

120

140

160

180

200

0 100pM 100nM 10μΜ

F5

F5/NP

Β

NP68

020406080

100120140160180200

0 100pM 100nM 10μΜ

F5

F5/NP

Α

NP68

c pm

(x1

0-3)

c pm

(x1

0-3)

F5 w/o

F5 +NP68F5/NP w/o

F5/NP +NP68

Γ

IL-2Rα

F5/NP CD8 T cells are hypoproliferativeto the antigen (anergy)

+IL-2

....but readily kill antigen-loaded cells in vivo (split tolerance)

B10F5 F5/NP

CFSElo:CFSEhi 0.93 2.0 13.1

Antigen-specific killing 0% 45% 95%

CFSE

lo hi

lo: w/o

hi: +NP68Principle of assay:

Lack of co-stimulation results to altered signaling cascade

Data based mainly on in vitro tolerized CD4 T cells

New partners of NFAT in anergic T cells

perpetuate the tolerant phenotype

•Differences (not many though...)

are observed in transcription profile

between optimally activated and

tolerant T cells

•Egr2 and Egr3 (but not the

“normal” Egr1) promote

transcription of other anergy-related

genes

•Antigen withdrawal in in vivo

tolerized cells results in fast reversal

of anergy

Other common alterations in anergic T cells

Internalization and downregulation of TCR

Downregulation of co-receptor (CD4 or

CD8)

Defective ZAP-70 activation

Constitutive TCRζ partial Y-ation

Increased steady-state levels of Ca2+ but

decreased Ca2+ response (desensitization)

Not necessarily all-in-one.....

T cell tolerance can be partial eg failure to proliferate

but ability to kill remains

The role of E3-ubiquitin ligases in T cell anergy

•Cbl-b-/- mice develop spontaneous (or MBP-

inducible) autoimmunity with auto-Ab production,

tissue infiltration by activated B & T cells and

parenchymal damage

•CD8.Cbl-b-/- T cells efficiently eradicate lymphoma

cells in mice

•Cbl-b-/- T cells: ↑proliferation, ↑ IL-2 production,

high levels of surface TCR/CD3, refractory to

tolerance induction

Data point towards an impairment of CD28 signaling

through Cbl-b function

The role of E3-ubiquitin ligases in T cell anergy

•T cells overexpressing GRAIL: ↓ proliferation, ↓ IL-2 production

•Dominant negative GRAIL in T cells: impaired anergy induction

•GRAIL upregulation in CD4 T cells is associated with remission in

patients with ulcerative colitis

•Mediates its action through sequestration of Ras away from the

membrane

The role of E3-ubiquitin ligases in T cell anergy

•Itch mutants: lymphohyperplasia and constant itching!

•Itch-/- CD4 T cells skewed towards Th2 (due to JUNB stability)

The role of E3-ubiquitin ligases in T cell anergy

•Roquin-mutant (sanroque) mice : increased susceptibility to

diabetes and lupus-like disease

•Marked increased in follicular-helper T cells, TFH, resulting in

excessive number of germinal centers

•Roquin negatively regulates expression of the co-stimulatory

molecule ICOS

Vav

MAPK/ERK activation

JunP

UbUbUb

RhoGTP

E3 ubiquitin ligases and signaling in anergic T lymphocytes

X

Negative costimulation and survival: important determinants of tolerance and autoimmunity

Pten+/- B & T cells are resistant to Fas-mediated apoptosis

4. BTLA-/- mice show no gross perturbations in lymphocyte development, cell activation or the composition of lymphoid organs.However, T cells from BTLA-deficient mice are hyperresponsive to TCR-induced proliferation in vitro. In addition, BTLA-deficient mice have greater severity and duration of experimental autoimmune encephalomyelitis, prolonged airway inflammation and increased rejection of minor mismatched allografts, further supporting a negative regulatory role for BTLA in vivo

1.

2.

3.

“Strong” phenotype in CTLA-4-/- mice but not classical autoimmune

siRNA downregulation of CTLA-4 ➱ faster T1D in NOD mice whereas

constitutive CTLA-4 ligation delays T1D in NOD mice

CTLA-4 mutations in humans are correlated with T1D, autoim.

hypothyroidism, Grave’s disease and increased rates of transplants

rejection

CTLA-4 is the strongest and best studied negative costimulator

Proposed modes of function for CTLA-4

CTLA-4 has a 50-fold higher affinity for B7.1,-2

CTLA-4 recruits Y-phosphatases (SHP2 & PP2A) resulting to reduced LAT -ation & ERK-activ.

Leads to reduced availability of key

components for TCR signaling

A novel mechanism for CTLA-4 function (in recently activated T cells)

TCR ligation reduces T cell motility (“stop signal”) to form effective synapses

CTLA-4 -/- T cells exhibit reduced motility and longer contacts with APCs-Ag in

vitro and in vivo (LN, 2 photon microsc.)

Prolonged contacts with APCs may lower thresholds for activation by self-Ags

and promote autoimmunity in CTLA-4-/- mice

CTLA4 wtCTLA4 -/-

moti

lity (

μm

3/m

in)

PD-1 and its ligands are negative costimulators involved in T cell tolerance

PD-L1: Immune cells, many tissues

PD-L2: Some immune cells, few tissues

Mild autoimmunity in PD-1 -/-mice in some strains (B6: glomerulonephritis, Balb/c:

dilated cardiomyopathy due to autoAb production, NOD mice: increased diabetes

incidence)

PD-1 polymorhisms in SLE, RA, MS and T1D patients

Some infectious agents (and some tumors) “use” PD-1 pathway to evade immune

response→”exhausted” CD8 T cells, chronic infections. Blocking PD-1 can result in

pathogen clearance

Distribution of their ligands suggest:

CTLA-4 may be important for tolerization in

2ndary lymphoid organs whereas PD-1 may be

critical for abrogating responses against

tissues

Mature DCs promote immunity-”Immature” DCs promote tolerance

Effector T cells Memory T cells

Activated T cells(not effectors)

Anergic T cells

Can we speak about “professional” tolerogenic APCs?

ArginaseIDO

Again: Not all in one...

Control of T cell responsiveness by DCs

TissueSelf Ag

APC

Pathogen

Self Ag specific TCR

Pathogen specific TCR

Proliferation Proliferation

Peripheral CD8+ T cell tolerance to self-Ags is regulated proximally to the TCR (and is not a global property of the T cell)

~10-20% of peripheral T cells bear two distinct TCRs

due to inefficient allelic exclusion in TCRa locus

Encounter with self Ag makes T cell tolerant

It can still respond to pathogen

Response to pathogen rescues function of tolerized TCR

Consequences (potential): autoimmunity, reaction against tumor Ags

Tissue damage

TissueSelf Ag

Anti-inflammatory neuropeptides (produced by immune cells,

too) promote immune tolerance

Effects of VIP and a-MSH in immune cell homeostasis and cytokine balance

Most of their action is due to increased cAMP levels and reduced NF-κΒ activation

Overview of B cell tolerance

Central tolerance of autoreactive B cells is mediated by receptor editing or clonal deletion in the bone

marrow

B cell anergy as a mechanism of peripheral tolerance

MD4 mice express HEL specific IgM & IgD (BCR)

ML5 mice express soluble HEL

MD4 X ML5

•Normal differentiation in BM but:

•Increased #T3 but decreased #follicular mature B cells

•Anergic phenotype: Low Abs in serum after HEL immunization

•Failure to differentiate to plasma cells in response to TLR ligands

The HEL model

Defective B cell anergy in

(MD4XML5) Cbl-/-Cbl-b-/- mice.

A role for ubiq. ligases in B cell

tolerance

B cell anergy as a mechanism of peripheral tolerance The Smith Ag model

•Sm is a nuclear self-Ag (component of snRNPs)

•Can activate B cells through BCR and TLR7

•Sm-specific B cells are related to pathology in some SLE patients and are present in MRL.lpr mice

•VH2-12 X i mice: No spontanous secretion of autoAbs but immunization with snRNPs induce them

•VH2-12 X Vk8 No secretion either spontanously or after immunization

Immunization overcomes tolerance only in some B cell specificities

Different levels/modes of anergy = f (specificity)

Nat. occurring anergic B cells exhibit a T3 phenotype (not transitional)

Highly enriched in autospecific clones

Very short lived compared to naïve B cells

Found in spleen and blood and LN

Estimated that 50% of immature B cells are destined to be anergic!

…but only ~6% of mouse B cells are anergic at a given moment

The signature of the anergic B cell?

Reduce

d r

esp

onse

to B

AFF

Ca2+

Altered signaling in anergic B cells due to prolonged Ag exposure

Bim

Understanding and manipulating tolerance mechanisms

may lead to a wide array of therapeutic possibilities

For reading:

1. T cell tolerance: Central and Peripheral. Cold Spring Harbor Persp Biol 2012;4:a006957

2. Peripheral tolerance in CD8+ T cells . Cytokines 46: 147-159 (2009)

3. The reverse stop-signal model for CTLA4 function Nat. Rev Immunol. 8: 153-160 (2008)

4. PD-1 and its ligands in tolerance and immunity Ann. Rev. Immunol. 677-704 (2008)

5. Mechanisms of tolerance Immunol. Reviews 241: 5-19 (2011)

6. Creating immune privilege: active local suppression that benefits friends, but protects foes. Nat. Rev Immunol. 8: 74-80 (2008)

7. Peripheral CD8 T cell tolerance to self-proteins is regulated proximally at the T cell receptor Immunity 28: 662-674 (2008)

8. B-cell anergy: from transgenic models to naturally occurring anergic B cells? Nat. Rev Immunol. 7: 633-643 (2007)

9. Physiologic regulation of central and peripheral T cell tolerance: lessons for therapeutic applications J. Mol. Med. 84: 887-889 (2006)

10. Regulation of immune tolerance by anti-inflammatory neuropeptides Nat. Rev. Immunol. 7: 52-63 (2007)

11. Mechanisms mantaining peripheral tolerance Nat. immunol. 11: 21-27 (2010)

12. Molecular mechanisms of CD4+T-cell anergy. Nat. Rev Immunol. 7: 599-609 (2007)