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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)