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• T-cell receptor signalling
• Toll-like receptor signalling
SIGNAL TRASDUCTION
determines
CELL RESPONSE
?
Signal Transduction in Lymphocytes
 Cytokine Receptor Signaling Pathways
 Chemokine Receptor Signaling Pathways
 Antigen-mediated Signaling Pathways
 TCR Signaling
 BCR Signaling
Adaptive Immunity
 NKR Signaling

TLR Signaling
Innate Immunity
Apoptotic Pathways
 Extrinsic stimuli- Fas-FasL, TNFR
 Intrinsic stimuli- Stress-DNA damage, UV, growth factor
starvation
Toll-like receptors
Immunity is about Recognition
of Danger….
How Immunity Evolved
• The immune system evolved under selective pressure imposed
by infectious microorganisms.
• Multicellular organisms have developed defense mechanisms
that are triggered by infection and protect the host by
destroying the invading microbes and neutralizing their
virulence factors.
How the Immune System
Recognizes Pathogens
immune
system
RESPONSE:
innate
adaptive
RECEPTORS:
dozens
millions
DIVERSITY:
germline
encoded
somatic gene
rearrangement
Strategies of the Innate Immunity
1.
2.
Recogniction of “microbical non-self”:
PAMPS and PPR
Recogniction of “missing self”
3.
Recogniction of “induced or altered self”
this concept relies on the detection of “markers on normal
self” and was proposed to explain why NK cells preferrentially killed
cells that express few or no MHC class I proteins on the cell surface.
this third strategy is based on the detection of markers of abnormal
self that are induced upon infection (viral infection) and cellular
trasformtion. Markers of abnormal self tag the affected cells for
elimination by the immune system.
Medzhitov and Janeway, Science (2002) vol. 296: 298-300
Recogniction of microbial non-self
•
Not only is it difficult to recognize pathogens because of their enormous
variability and molecular heterogeneity, but microorganisms have an
extremely high mutation rate
•
Good targets for recognition by the innate immune system:
1) are shared by a large number of pathogens and are therefore patterns, and not
simply molecular structures
2) must be conserved products of microbial metabolism that do not undergo
antigenic variation, and are essential to the survival of the organism
3) structures must the absolutely distinct from self antigens
These structures are called Pathogen-Associated Molecular Patterns
(PAMPs)
Pattern Recognition Receptors
(PRRs)
•
The principal functions of pattern recognition receptors that recognize PAMPs
include opsonization, activation of complement and coagulation cascades,
phagocytosis, activation of proinflammatory signaling pathways and induction
of apoptosis
•
In addition, PRRs are a link between innate and adaptive immunity: uptake of
the pathogen expressing the PAMPs by APCs can lead to the induction of
MHC-peptide molecules as well as costimulatory molecules in order to
activate the adaptive immune response
•
The main difference between PRRs and clonally distributed receptors on Bcells and T-cells is that they are germline-encoded
•
Pattern recognition receptors can be expressed on the cell surface, in
intracellular compartments, or secreted into the bloodstream and tissue fluids
Enter the Toll-like receptor family….
• They are PRRs expressed on the cell surface
• Important for the detetion of microrganisms
by the innate immune system and triggering
of inflammatory response
• Play a role in activating the adaptive
immune response
Immunity at the
Host/Environment Interface
gram- bacteria
innate
gram+ bacteria
yeasts
MØ
APC
viruses
neutrophils
adaptive
T cell
parasites
patterns
pattern
recognition
receptors
surface receptors
effector molecules
counterreceptors
Antigen presenting cells
• Dendritic cells
• Macrophages
• B cells
Up-take
degli Ag
Superfici
e
epiteliale
A
g
MF
NK
Cellule T
CTL,
helper
e
regolatori
e
DC immatura
Precursore
E
O
Citochi
ne
Migrazion
e
Maturazio
ne
Presentazione
degli Ag
Selezio
ne dei
linfociti
DC
matura
Organi
linfoidi
Enter the Toll Receptor Family…
• The Toll protein controls embryonic development in
Drosophila, and is required for antifungal immune responses
in adult flies.
• Toll is a type I transmembrane receptor. The extra-cellular
domain contains leucine-rich repeats. The cytoplasmic domain
is similar to that of IL-1 receptor.
• Ten human homologues of Toll (Toll-like receptors, TLRs)
have been identified.
• The signaling pathway of the TLR family leads to activation of
NF-kB through an adaptor protein, MyD88, and IL-1Rassociated kinase (IRAK).
TLRs and Their Ligands
lipoproteins (di-/triacylated)
lipoarabinomannan
nucleotides
LPS (leptospira)
LPS (Gram -)
nucleotide analogs
LPS (P. gengivalis)
taxol (plants)
PGN (Gram +)
F protein (RSV)
unmethylated anti-viral
zymosan
(yeast)
Hsp60 (host)
compounds
CpG DNA
flagellin
GPI
anchor
(T.
cruzi)
fibronectin (host)
ds RNA
MD-2
TLR7
TLR4
TLR6
TLR2
TLR1
TLR5
TLR9 TLR3
The Localization of TLRs
TLR3
TLR2
TLR4
Nod2
endoplasmic
reticulum
GP96
TLR7
endosomal
compartment
intestinal lumen
TLR9
TLR5
Structure
TLRs are type I
transmembrane proteins:
• Extracellular domain
contains leucine-rich
repeats
• Cytoplasmic Toll/IL-1
receptor (TIR) domain
Signal transduction
TLR signaling pathways differ from one another and elicit
different biological responses.
2 pathways:
• MyD88 – dependent pathway: used by all TLRs
• MyD88 – independent pathway: TLR4 and TLR3
MyD88-dependent signaling pathway
•
MyD88:
–
–
adaptor molecule that associates with
the TIR domain to TLR TIR domain
recruits the kinase IRAK to the TLR
after stimulation of TLR by ligands
 IRAK:
 Ser/thr kinase
 becomes phosphorylated during

recruitment to the receptor complex
associates with TRAF6 after
phosphorylation
 TRAF6:

Induces activation of TAK1
 TAK1:
 Activates via NIK and IKK NF-kB
Akira, Curr.Op.Immunol. 15: 5, 2003
19
Focus onTLR4
• TLR4 ligand
• LPS binding complex
TLR4 LIGAND
• Non self ligands
– LPS ( Gram-negative
bacteria)
– Taxol (plants)
– Viable Mycobacterium
tubercolosis bacilli
– RSV fusion protein
– Envelope proteins from
mouse mammary tumor
virus or from murine
leukemia virus
• Endogenous ligands
• Mouse -defensin
• HSP60 (Clamidia and
human)
• HSP70
• Fibronectin domain A
• Satureted fatty acid
• Minimally modifued LDL
Lipopolysaccharide
LPS recogniction complex
•
•
•
•
LBP, a 60 kDa serum glycoprotein that
binds to the lipid A moiety of LPS, is a lipid
transferase catalyzing LPS transfer to outer
membrane to CD14
CD14, a glycoprotein expressed on
myelomonocytic cells. Mice with a target
deletion of the gene encoding CD14 are
hyporesponsive to LPS. CD14 seems to have
a role in amplifyng LPS responses.
TLR4
MD-2, is an indispensable molecule for LPS
responses. A variety of in vitro studies
demostrated that LPS hyporesponsiveness in
cells expressing TLR4 alone or TLR4 with
mutant MD-2 was rescued bt the transfection
of MD-2 cDNA or soluble MD-2 protein.
Four adaptors for TLR4
MyD88 and TIRAP take part in TLR4mediated proinflammatory cytokine
production and B cell activation, but
not in induction of the gene
encoding IFN- and IFN-inducible
genes, the so-called MyD88independent pathway17, 18, 21, 22.
TRIF is essential in the MyD88independent pathway shared by
TLR3 and TLR4 (refs. 25,26).
TRAM specifically participates in
the MyD88-independent pathway of
TLR4, but not TLR3, signaling.
FOCUS on TLR9
• TLR9 ligand: CpG DNA:
Bacterial DNA triggers cells of the innate immune system to proliferate
and become functionally active. This immune stimulation is mediated
by unmethylated CpG motifs present with high frequency in bacterial
DNA, but rare in mammalian DNA. The responsiveness to CpG DNA
is completely lost in MyD88 KO and, obviously, in TLR9 KO mice.
Also in humans, CpG recogniction is mediated by TLR9.
•
TLR9 subcellular localization: it is expressed on endosomial
vescicles
• TLR9 signaling pathway
Scheme of subcellular
localization of CpG ODN and
TLR9
Under physiologic conditions, TLR9
molecules are located below the
plasma membrane, presumably in
vesicles. Cy3-labeled CpG ODN
associate with the cell surface and are
transported to these vesicles within
10 min. By 2 h the size and number
of CpG ODN-containing endosomes
increases significanty, and they
relocate from just below the plasma
membrane to intracellular regions. In
some cases, the CpG ODN and TLR9
molecules co-localize within the same
vesicles, recruit MyD88, and initiate
the signal transduction process.
Takeshita et al. Sem.in Imm. 16 (2004) 17-22
Attivazione delle cellule T
SIGNAL 2
APC-T cell adhesion
SIGNAL 1
MHC:pep - TCR
Cytokine triggers
Receptor
CO-STIMULATION
IL-2
IL-2R
G0
Cytokine
Promoter
NUCLEUS
IL-2
Cytokine
Production
G1
S
M
G2
PROLIFERATION
DIFFERATION
Function of costimulators in
T cell activation
Initial contact
Stabilization
Naive T:DC unique
Janeway 5th
Edn
All steps Ag non-specific. All common, transient
Stability comes from?
Ag alone?
Costimulation alone?
Ag plus costimulation?
Il complesso del TCR
Il TCR è una glicoproteina eterodimerica costituita, nella maggior parte dei
linfociti T circolanti, da due diverse catene chiamate a e .
3 domini:
1.
La porzione extracellulare di ogni catena è formata da una regione
COSTANTE (C) e da una regione VARIABILE (V) e da una corta regione
CERNIERA (HINGE) che contiene i residui di cisteina tra cui si realizza il
ponte disolfuro intercatena
2.
Un dominio transmembrana, dove sono presenti AA carichi positivamente
che interagiscono con le altre molecole di membrana
3.
Un corto dominio intracitoplasmaticco rispettivamente di 9 e 12 AA per la
catena a e .
Le code intracellulari delle catene a e  sono troppo corte per riuscire a trasdurre
il segnale proveniente dall’ingaggio del TCR. Questa funzione viene svolta
da un complesso multiproteico di membrana detto CD3 stabilmente
associato al TCR.
Il complesso CD3 è costituito da tre proteine, omologhe tra loro, dette g,d e e
che si associano a formare degli eterodimeri g:e e e:d. Il complesso CD3 è
costituito da altre due catene chiamate z e h, le quali si associano tra loro
a formare l’omodimero z-z (80% dei casi) o l’eterodimero z-h.
Il complesso del TCR (2): Sequenze ITAM
(immunoreceptor-based tyrosine activation-motif)
La porzione citoplasmatica di ciascun componente del complesso CD3
e la catena z del TCR contengono un motivo unico detto ITAM
(immunoreceptor-based activation motif) costituito dalla sequenza
consenso:
YxxL/V X(6-8) YxxL/V
Le sequenze ITAM sono necessarie e sufficienti per l’attivazione
indotto dall’ingaggio del TCR.
I residui di Tyr all’interno delle sequenze ITAM vengono rapidamente
fosforilate in seguito all’ingaggio del TCR e forniscono dei siti di attacco
per il reclutamento in membrana per proteine contenenti motivi SH2.
IL PRIMO SEGNALE INTRACELLULARE CHE INDICA CHE I
LINFOCITI HANNO RICONOSCIUTO IL LORO ANTIGENE
SPECIFICO è LA FOSFORILAZIONE DELLE TYR NELLE ITAM
TCR molecular signalling
TCR
CD8
a

CD3
d
-S-S-
e
Va
V
Ca
C
C
x
x
C
Lck
g
e
z
z/h
-S-S-
P
P
p56lck
p59fyn
Regulation of Src Tyr-Kinase family
C
x
C
CD8
CD4
CD3
-S-S-
P
p56lck
CD4
Phosphorylatio
n of activating
tyrosine
Regione
unica
CD45
Dominio SH3
Dominio SH2
Tyr attivante
Dominio
chinasico
Tyr inibitoria
Phosphorylation of
inhibitory tyrosine
residue leads to a
conformational
change that blocks
the active site
Csk
TCR molecular signalling
TCR
CD8
a

-S-S-
LcK
CD3
d
e
Va
V
Ca
C
-S-S-
CD4
CD3
g
e
z
z/h
-S-S-
LcK
Fyn
ON
ZAP70
Una volta che le ITAM delle code
citoplasmatiche del recettore sono state
fosforilate, esse reclutano in membrana la
protein-tyr chinasi ZAP70 che viene attivata in
seguito a fosforilazione da parte della chinasi
LcK
Tyrosin Kinase : ZAP/SYK family
SH2 SH2
Y
Kinase
TCR molecular signalling
TCR
CD3
d
e
Va
V
Ca
C
-S-S-
CD3
g
e
z
z/h
LAT
-S-S-
p59fyn
PLCg
ZAP70
LAT (linker for activation of T cells) è un cosidetto ADATTATORE: contiene una corta sequenza
extracellulare, un dominio transmembrana e un ampio dominio citoplasmatico contenente 9
residui di tyr altamente conservati. È associata alla faccia interna della membrana plasmatica
mediante residui di cisteina che sono “palmitoilati”.
L’associazione di LAT è essenziale per la propagazione del segnale: l’attivazione di alcuni fattori
trascrizionali e la produzione di IL-2 non si verifica in cellule T che mancano di LAT.
Altered peptide ligands: APL
I peptidi antagonisti inducono una subottimale fosforilazione delle subunita z del
CD3 con conseguente assenza di richiamo
di ZAP 70
Questo può portare ad anergia delle
cellule T
Attivazione di NFAT
CD3
e
z
z/h
LAT
-S-S-
PIP2
DAG
ICRAC
IP3
PLCg
[Ca2+]
ZAP70
CALCINEURINA
Ca2+
RE
[Ca2+]
NFAT ON
NFAT
NFAT
OFF
Attivazione della PKC-q
TCR complex
PLC-g
DAG
PKC-q
IP3
Lck, Fyn, ZAP70
LAT
•Incremento [Ca]
intracellulare
IKKg
SEK 1
JNK
IkB
NFkB
•Attivazione calcineurina
•ON NFAT family
IKK IKKa
DEGRADAZION
E
PROTEOSOMA
26S
Attivazione
AP1
NUCLEO
La costimolazione amplifica il
segnalling del TCR e stabilizza il
messaggero per l’IL-2
La costimolazione amplifica il
segnalling del TCR e stabilizza il
messaggero per l’IL-2
E’ necessario che almeno tre TCR
siano attivati sulla sulla superficie
cellulare per avere un segnale
all’interno della cellula
In seguito ad attivazione la cellule T down-regola il proprio TCR
Nel caso in cui una cellula T esprima due TCR, solo il TCR interessato
nel legame con il complesso MHC+pep è downregolato
Un singolo complesso MHC+peptide puo serialmente attivare
molteplici TCR: serial triggering
Costimulation
Multiple families of costimulatory molecules
APC
T cell
B7 : B7.1 and B7.2 (CD80, CD86) binds CD28 on T cell
(restricted to lymphoid tissues, primarily “activn of naïve T”?)
B7-H2,
ICOS
B7-H3
???
(both seen widely expressed on non-lymphoid tissues,
ie epithelium, primarily interacting with effectors?
Not affected by CTLA4)
B7 H3 selectively downregulates Th1 activity, favoring Th2: 2003
OX40
CD4
OX40L
4-1BB
CD8
4-1BBL
(mount normal CD4 or CD8 primary responses,
but poorer secondary responses.)
Multiple families of costimulatory molecules
APC
T cell
B7 : B7.1 and B7.2 (CD80, CD86) binds CD28 on T cell
Disruption leads to very weak immune responses ~10% of normal.
Thus “the” major co-stimulatory family for initiating T cell immunity
B7-H2,
ICOS
B7-H3
???
Ox40 (CD4)
OX40L
4-1BB (CD8)
4-1BBL
Disruption results in subtle effects,
such as attenuated or different secondary responses;
different types of response (Th1 vs Th2 bias).
Bottom line: Multiple families of costimulatory molecules
are found to direct nuances of immune responses: sustaining or
differentiating immune responses.
Nuance: IgM vs IgE to parasite, so not trivial
SINAPSI
IMMUNOLOGICA
Sull’APC, quando il TCR
Riconosce il complesso
MHC-peptide, numerose
proteine di membrana
e intracellulari vengono
mobilizzate rapidamente.
La regione di contatto
fisico tra APC e cellula T
È chiamata SINAPSI
IMMUNOLOGICA o
CLUSTER di ATTIVAZIONE SOPRAMOLECOLARE (SMAC).
TCR, CD3, catene z , i
corecettori CD4 o CD8, e
il CD28 sono mobilizzati
al centro della sinapsi.
Le integrine restano
invece alla sua periferia.
cSMAC= central
Supramolecular
Cluster
pSMAC= periferal
Supramolecular
Cluster
Analisi in “imaging al microscopio confocale ”
della formazione della sinapsi immunologica
Una cell.T specifica per un determinato complesso MHC-peptide lega un’APC.
CD3 (verde) si localizza sullo cSMAC.
L’integrina LFA-1 (rosso) invece si localizza sullo pSMAC.
CD3 è al centro e LFA-1 è alla periferia della sinapsi immunologica.
B7.1 e B7.2: omodimeri, membri della superfamiglia delle Ig
se trasfettate in fibroblasti conferiscono capacità costimolatoria
CD28. Glicoproteina espressa costitutivamente sulla
superficie dell’80% delle cellule T umane (tutte le CD4 e il
50% delle CD8) e sul 100% delle cellule T murine.
e’ upregolato in seguito ad attivazione.
Inibizione della attività citotossica mediata da
anticorpi bloccanti anti-B7.1 e anti B7.2
Priming delle cellule T CD4 anti-OVA in vitro da parte di APC
KO per B7.1 e B7.2
Priming con APC dKOB7 in
presenza delle citochine indicate,
ristimolazione in presenza di APC
wt
Priming e ristimolazione con le APC dKO B7
CTLA4: è upregolato in seguito ad attivazione delle cellule T
ma con una cinetica diversa rispetto al CD28. E’ upregolato 48h
dopo l’attivazione e viene di nuovo downregolato 96h dopo.
Lega B7.1 e B7.2 con una affinità molto superiore al CD28. Ha
un ruolo negativo
CTLA4 -- the essential off signal
• CTLA4, expressed by T cells upon activation,
competes with CD80 or 86 for CD28.
• Relevance to immune regulation
• -/-: T cell hyperproliferation, cancer,death
• elevated CTLA4: induction of tolerance?
Co-segnali inibitori
Cellule T
Cellule presentanti l’antigene
B7.1 (CD80)
B7.2 (CD86)
CO-STIMOLATORIE
CD28
CTLA4 (CD152)
CO-INIBITORE
CTLAA4 lega le molecole B7 con un’affinità
circo 20 volte superiore a CD28 e invia segnali
inibitori alle cellule T attivate: ciò a si che la
progenie attivata di una cellula T naive sia meno
sensibile alla stimolazione da parte delle APC e
limita la quantità di IL-2 secreta.
Il legame CTL4-B7 è essenziale per limitare la
risposta proliferativa delle cell.T all’Ag e e a
B7.2. Questo è stato confermato generando
topi con CTLA4 difettivo: tali topi sviluppano un
disordine letale caratterizzato da proliferazione
linfocitaria ecessiva.
Il meccanismo molecolare attraverso cui
CTLA-4 inibisce l’attivazione delle cellule T
non è ancora stato elucidato.
È stato dimostrato che il legame di CTLA4
è in grado di indurre anergia/tolleranza nei
linfociti T periferci.
È ipotizzabile che esso agisca inibendo la
progressione nel ciclo cellulare e
inducendo la produzione di TNF-.
Th1/Th2 basics...
• Mosmann/Coffman ‘86: CD4 T clones do not exhibit random patterns of cytokine
gene expression following Ag stimulation
• Terminology
• Th0: Ag-specific, activated CD4 T cell that has not yet committed to a Th1 or
Th2 biased pattern of cytokine gene expression.
• Th1: IFNg, TNF (not IL-2)
• Th2: IL-4,IL-5,IL-13
• Th3: TGFb, important in mucosal immunity, low dose tolerance (ie to food)
Naïve Th Cells Can Differentiate
Into Th1 or Th2 Cells
Naive Th cells
Short-term
stimulation
Chronic
stimulation
Th1
cell
Long term
Memory cells
IFNγ
IL-2
ThP
ThO
ThM
cell
cell
cell
Th2
cell
IL-2
IFNγ
IL-2
IL-4
IL-5
IL-10
IL-4
IL-5
IL-6
IL-10
IL-2
Functions of Th1 and Th2 Cells
Th1
Th2
cell
cell
Inhibits production
IL-10
IFNγ
IL-4 IL-5
Activates
Activates
Macrophage
Mast cell
B cell
Eosinophil
Antibodies (including IgE)
Functions of Th1 and Th2 Cells
Le cellule Th1 possono anche avere una funzione
citotossica che si esplica attraverso
Il segnale FAS-FASL
Sistemi alternativi per la generazione di Th1 e
Th2
Figure 10-13
Figure 10-13 part 1 of 2
Figure 10-13 part 2 of 2