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 tolerance
Regulatory 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

Tolerance in immune-privileged sites

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 (TGF)

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

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

A rough outline of lymphocyte tolerance

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

Negative selection: survival of the fittest

YOU ARE HERE

Useless Neglect Useful Harmful Positively select Negatively select

Number of cells

Low Affinity of TcR/MHC interaction

High

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

AIRE: AutoImmune REgulator APECED (Autoim. PolyEndocrinoPathy Candidiasis Ectodermal Dystrophy)

Ocasionally potentially autoreactive naive T cells escape in the periphery Thymus

activation T T T T
uptake

Periphery

T T T effector T T T cells T

release

mTEC

DC

parenchymal cell

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
TCR-tg T cells
1. Activation of Th cell lines only through TCR.
Anergy reversible by IL-2. Stimulus withdrawal does not reverse anergy. 2. Adaptive tolerance Anergy irreversible by IL-2 Ag withdrawal reverses anergic phenotype Similar anergic phenotypes in TCR x self-Ag tg models

Ag

#
Time

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!!

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

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

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

A
F5
26.1%

F5/NP
5.4%

F5/Rag1-/MFI:454

F5/NP/Rag1-/MFI:544 85.7%

34.8%

CD8 CD44

V11
MFI:91 16.2% MFI:120 37.9%

CD69

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

200 180 160 140 120 100 80 60 40 20 0

F5 F5/NP

+IL-2
200 180 160 140 120 100 80 60 40 20 0

F5 F5/NP

100pM

100nM

10

100pM

100nM

10

NP68

NP68

F5 +NP68 F5 w/o F5/NP w/o

F5/NP +NP68

IL-2R

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

B10

F5

F5/NP

CFSElo:CFSEhi

0.93

2.0 45%

13.1 95%

Antigen-specific killing 0%

Principle of assay:

lo

lo: w/o hi: +NP68 hi

CFSE

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 TCR 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-/cells in mice

T cells efficiently eradicate lymphoma

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 ICOS

Roquin negatively regulates expression of the co-stimulatory molecule

E3 ubiquitin ligases and signaling in anergic T lymphocytes

UbUbUb

Vav

GTP

Rho

MAPK/ERK activation
P

Jun

Negative costimulation and survival: important determinants of tolerance and autoimmunity

1.

2.

3.

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

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

CTLA-4 is the strongest and best studied negative costimulator

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, Graves disease and increased rates of transplants rejection

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)

CTLA4 -/- CTLA4 wt

motility (m3/min)

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

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 responseexhausted 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?

Again: Not all in one...

IDO

Arginase

Control of T cell responsiveness by DCs

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
Tissue Self Ag
Tissue Self Ag

Self Ag specific TCR

Proliferation

Proliferation

Tissue damage

Pathogen specific TCR

APC

Pathogen
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

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- 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

The HEL model

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

Defective B cell anergy in (MD4XML5) Cbl-/-Cbl-b-/- mice. A role for ubiq. ligases in B cell tolerance

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

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

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 nave 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?

Altered signaling in anergic B cells due to prolonged Ag exposure

Ca2+

Bim

Reduced response to BAFF

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)