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SEMINARIO
Exosomi
Risposta immunitaria
Vescicole di membrana come vettori di risposte immunitarie
Porzioni della membrana plasmatica di cellule coinvolte nella risposta immunitaria
possono essere trasferite fra cellule, sia tramite contatto diretto (mediante i processi
recentemente descritti di «nibbling» (rosicchiamento), trogocitosi e nanotubi) che tramite
la secrezione di vescicole di membrana.
Le conseguenze funzionali di tali trasferimenti includono l’induzione, amplificazione e/o
modulazione delle risposte immunitarie nonché l’acquisizione di nuove proprietà
funzionali da parte delle cellule che le ricevono, quali capacità migratorie o metastatiche.
Inoltre, nelle vescicole di membrana secrete sono stati identificati mRNAs e microRNAs, e
ciò ha sollevato l’eccitante ipotesi che il trasferimento di materiale genetico potesse
influenzare il comportamento delle cellule riceventi.
Complessivamente, tali dati portano all’ipotesi che il trasferimento di membrane sia un
modo comune di comunicazione intercellulare.
Théry C, Ostrowski M, Segura E. Membrane vesicles as conveyors of immune responses. Nat Rev Immunol. 2009 Aug;9(8):581-93.
Definizioni (vedi figura Davis)
«Nibbling» (rosicchiamento): Capacità che hanno le cellule dendritiche di
strappare fisicamente frammenti di membrana da cellule vice durante un
contatto stretto senza indurre la morte della cellula donatrice.
Trogocitosi: Trasferimento di frammenti della membrana plasmatica da una
cellula ad un’altra senza indurre la morte cellulare. Questo processo è mediato da
segnalamento mediato da recettore in seguito a contatto cellula-cellula.
Nanotubi: Canale membranoso di 50-200 nm di diametro che collega cellule per
lunghe distanze.
Vescicole di membrana: Struttura sferica o approssimativamente sferiche limitata
da un bilayer lipidico che racchiude un carico solubile.
Théry C, Ostrowski M, Segura E. Membrane vesicles as conveyors of immune responses. Nat Rev Immunol. 2009 Aug;9(8):581-93.
Meccanismi per il
trasferimento intercellulare di
proteine di membrana tra
cellule immunitarie
(Fig.1)
Davis DM. Intercellular transfer of cell-surface proteins is common and can affect many stages of an immune response. Nat Rev Immunol. 2007 Mar;7(3):238-43.
Didascalia Fig. 1 Davis -I
a | Le proteine potrebbero essere sradicate dale membrane delle cellule. Non esistono prove
sperimentali che ciò succeda, ma la valutazione dell’energia coinvolta indica che sia fattibile.
b | La scissione proteolitica potrebbe facilitare il trasferimento intercellulare di ectosmini proteici.
Ciò occorre per alcune protein note per muoversi fra cellule, quali la proteina indotta da stress MIC
(MHC-class-I-polypeptide-related sequence), che viene scissa alla superficie delle cellule tumorali e
si può legare e bloccare il suo recettore NKG2D (NK group 2, member D) su cellule T cells e cellule
“natural killer”.
c | Il trasferimento potrebbe essere mediato da corpi racchiusi da membrane, vescicole o organlli
di maggiori dimensioni nei punti di contatto intercellulare. Questo processo potrebbe coinvolgere
la secrezione di vescicole spacializzate quali gli exosomi. Tuttavia, dati riguardanti numerosi tipi di
cellule e protein indicano che vi è un processo particolarmente commune, con caratteristiche
distinte dalla secrezione di exosomi, che permette lo scambio intercellulare di protein di membrane,
detto trogocitosi. Le basi molecolari precise della trogocitosi non sono ancora chiare. Il processo
potrebbe coinvolgere, ad esempio, l’endocitosi delle due membrane di una sinapsi [ad es.
Immunologica] o forse una membrane chiusa su se stessa è strappata quando le cellule si separano.
Davis DM. Intercellular transfer of cell-surface proteins is common and can affect many stages of an immune response. Nat Rev Immunol. 2007 Mar;7(3):238-43.
Didascalia Fig. 1 Davis -II
d | La fusione intercellulare delle membrane potrebbe produrre piccolo ponti di membrane che
permettono il trasferimento di proteine. Ponti di membrane sono stati osservati in fotografie al
microscopio elettronico di sinapsi immunologiche di cellule T citotossiche, ma non ci sono prove che
questi ponti facilitino lo scambio di proteine fra le cellule. Coloranti del citosol di solito non vengono
trasferiti fra le cellule, e quindi deve esistere qualche sorta di blocco attraverso i ponti di membrane.
e | Nanotubi di membrane, forse derivati dalla fusion di membrane o da ponti di membrane nel
sito del contatto intercellulare, potrebbero facilitare il trasferimento di proteine fra cellule
distanti. Nanotubi di membrane sono comunemente osservati fra diversi tipi di cellule
immunologiche, ma la caratterizzazione del tipo di traffic fra le cellule immune lungo i nanotubi è
carente.
Davis DM. Intercellular transfer of cell-surface proteins is common and can affect many stages of an immune response. Nat Rev Immunol. 2007 Mar;7(3):238-43.
La sinapsi immunologica funge da piattaforma per
facilitare il passaggio di material genetico tra le
cellule
 Durante la formazione di una sinapsi immunologica, le
molecole coinvolte nel riconoscimento dell’antigene (ad
es. il ”T cell receptor; TCR) e le molecole del “peptideloaded major histocompatibility complex; pMHC) si
muovono verso un aggregato centrale circondato da un
anello periferico arricchito in molecole di adesione (ad
es. l’integrina “leukocyte function-associated antigen 1”
(LFA1) e le ”intercellular cell adhesion molecules; (ICAMs)
e di citoscheletro di actina.
 Il linfocito T orienta il suo “microtubule-organizing centre
(MTOC) e i compartimenti di secrezione (ad es. l’apparato
di Golgi e “i multivesicular bodies» (MVBs) verso la
“antigen presenting cell” (APC).
 Noi proponiamo che la sinapsi immunologica fornisce
una via di maggiore efficienza per lo scambio di materiale
genetico mediante la combinazione di differenti
meccanismi, incluso la secrezione polarizzata di exosomi
carichi di microRNA (miRNA), transendocitosi e ponti di
membrana. I patogeni, incluso batteri e virus, si
appropriano delle sinapsi biologiche per propagrasi da
cellula a cellula.
Mittelbrunn M, Sánchez-Madrid F. Intercellular communication: diverse structures for exchange of genetic information. Nat Rev Mol Cell Biol. 2012 Apr 18;13(5):328-35.
Processi in cui sono coinvolte
microvescicole
Presentatione di antigene
Produzione di «armed effector T cells» - 1
Per essere attivate, una cellula T “naïve” deve riconoscere un peptide estraneo legato ad
una molecola del complesso di maggiore istocompatibilità (MHC) “self”.
Ciò di per se tuttavia non è sufficiente per l’attivazione.
L’attivazione richiede una consegna simultanea di un segnale co-stimolatorio da una cellula
specializzata presentatrice di antigene.
Solo le cellule dendritiche, macrofagi e linfociti B sono in grado di esprimere sia le classi di
molecole MHC che le molecole co-stimolatorie, presenti sulla superficie cellulare, che
portano all’espansione clonale delle cellule T “naive” e al loro differenziamento in “armed
effector T cells”.
I più potenti attivatori delle cellule T “naive” sono le cellule dendritiche mature e si ritiene
che queste inizino la maggior parte, forse tutte, le risposte mediate da cellule T in vivo.
http://www.ncbi.nlm.nih.gov/books/NBK27118/
Produzione di «armed effector T cells» - 1
Le cellule dendritiche immature nei tessuti catturano antigeni nei siti di infezione
e vengono attivate per viaggiare fino al tessuto linfatico locale.
In questa sede maturano in cellule che esprimono elevati livelli di molecole costimolatorie e le molecole di adesione che mediano le interazioni con le cellule T
“naive” che continuamente recircolano attraverso questi tessuti.
L’attivazione e l’espansione clonale delle cellule T “naive” nel momento del loro
iniziale incontro con l’antigene presente sulla superficie di una cellula che
presenta l’antigene è speso designata “priming” (innesco), per distinguerla dalle
risposte delle cellule T effettrici “armate” all’antigene presente sulle loro cellule
bersaglio, e con le risposte delle cellule T memoria “primed”.
Antigen-presenting cells are distributed differentially in the lymph
node
Dendritic cells are found throughout the cortex of the lymph node in the
T-cell areas.
Macrophages are distributed throughout but are mainly found in the
marginal sinus, where the afferent lymph collects before percolating
through the lymphoid tissue, and also in the medullary cords, where the
efferent lymph collects before passing via the efferent lymphatics into
the blood.
B cells are found mainly in the follicles. The three types of antigenpresenting cell are thought to be adapted to present different types of
pathogen or products of pathogens, but mature dendritic cells are by far
the strongest activators of naive T cells.
http://www.ncbi.nlm.nih.gov/books/NBK27118/figure/A1023/?report=objectonly
Naive T cells encounter antigen during their recirculation through peripheral
lymphoid organs
Naive T cells recirculate through peripheral lymphoid organs, such as the
lymph node shown here, entering through specialized regions of vascular
endothelium called high endothelial venules. On leaving the blood vessel, the
T cells enter the deep cortex of the lymph node, where they encounter
mature dendritic cells. Those T cells shown in green do not encounter their
specific antigen. They receive a survival signal through their interaction with
self MHC:self peptide complexes and leave the lymph node through the
lymphatics to return to the circulation. T cells shown in blue encounter their
specific antigen on the surface of an antigen-presenting cell and are activated
to proliferate and to differentiate into armed effector T cells. These antigenspecific armed effector T cells, now increased a hundred-fold to a thousandfold
in number, also leave the lymph node via the efferent lymphatics and enter the
circulation.
http://www.ncbi.nlm.nih.gov/books/NBK27118/figure/A1025/?report=objectonly
Activation of naive T cells requires two independent signals
Binding of the peptide:MHC complex by the T-cell receptor
and, in this example, the CD4 co-receptor, transmits a signal
(arrow 1) to the T cell that antigen has been encountered.
Activation of naive T cells requires a second signal (arrow 2),
the co-stimulatory signal, to be delivered by the same
antigen-presenting cell.
http://www.ncbi.nlm.nih.gov/books/NBK27118/figure/A1034/?report=objectonly
The principal co-stimulatory molecules expressed on antigenpresenting cells are B7 molecules, which bind the T-cell protein
CD28
Binding of the T-cell receptor (TCR) and its co-receptor CD4 to the
peptide:MHC class II complex on the antigen-presenting cell (APC)
delivers a signal (arrow 1) that can induce the clonal expansion of
T cells only when the co-stimulatory signal (arrow 2) is given by
binding of CD28 to B7 molecules. Both CD28 and B7 molecules
are members of the immunoglobulin superfamily. B7.1 (CD80)
and B7.2 (CD86) are homo-dimers, each of whose chains has one
immunoglobulin V-like domain and one C-like domain. CD28 is a
disulfide-linked homodimer in which each chain has one V-like
domain.
http://www.ncbi.nlm.nih.gov/books/NBK27118/figure/A1035/?report=objectonly
The requirement for one cell to deliver both the antigenspecific signal and the co-stimulatory signal is crucial in
preventing immune responses to self antigens.
In the upper panels, a T cell recognizes a viral peptide on
the surface of an antigen-presenting cell and is activated
to proliferate and differentiate into an effector cell
capable of eliminating any virus-infected cell. However,
naive T cells that recognize antigen on cells that cannot
provide co-stimulation become anergic, as when a T cell
recognizes a self antigen expressed by an uninfected
epithelial cell (lower panels). This T cell does not
differentiate into an armed effector cell, and cannot be
stimulated further by an antigen-presenting cell
presenting that antigen.
http://www.ncbi.nlm.nih.gov/books/NBK27118/figure/A1038/?report=objectonly
Presentazione di antigene da parte delle cellule
dendritiche
Dendritic cells mature through at least two definable stages to become potent
antigen-presenting cells in lymphoid tissue
Dendritic cells arise from bone marrow progenitors and migrate via the blood to
peripheral tissues and organs, where they are highly phagocytic via receptors such
as DEC 205 and are actively macro-pinocytic but do not express co-stimulatory
molecules (top panel). At sites of infection they pick up antigen and are induced to
migrate via the afferent lymphatic vessels to the regional lymph node (see Fig.
8.15). Here they exhibit high levels of T-cell-activating potential but are no longer
phagocytic. Dendritic cells in lymphoid tissue express B7.1, B7.2, and high levels of
MHC class I and class II molecules, as well as high levels of the adhesion molecules
ICAM-1, ICAM-2, LFA-1, and LFA-3 (center panel). They also express high levels of
the dendritic-cell-specific adhesion molecule DC-SIGN, which binds ICAM-3 with
high affinity.
http://www.ncbi.nlm.nih.gov/books/NBK27118/figure/A1040/?report=objectonly
Presentazione di antigene da parte dei macrofagi
Microbial substances can induce co-stimulatory
activity in macrophages
If protein antigens are taken up and presented by
macrophages in the absence of bacterial components
that induce co-stimulatory activity in the macrophage, T
cells specific for the antigen will become anergic
(refractory to activation). Many bacteria induce the
expression of co-stimulators by antigen-presenting
cells, and macrophages presenting peptide antigens
derived by degradation of such bacteria can activate
naive T cells. When bacteria are mixed with protein
antigens, the protein antigens are rendered
immunogenic because the bacteria induce costimulatory B7 molecules in the antigen-presenting
cells. Such added bacteria act as adjuvants.
http://www.ncbi.nlm.nih.gov/books/NBK27118/figure/A1043/?report=objectonly
Presentazione di antigene da parte dei linfociti B
B cells can use their immunoglobulin receptor to present specific antigen very efficiently to T cells
Surface immunoglobulin allows B cells to bind and internalize specific antigen very efficiently. The internalized
antigen is processed in intracellular vesicles where it binds to MHC class II molecules. These vesicles are then
transported to the cell surface where the MHC class II:antigen complex can be recognized by T cells. When the
protein antigen is not recognized specifically by the B cell, its internalization is inefficient and only a low density
of fragments of such proteins are subsequently presented at the B-cell surface (not shown).
http://www.ncbi.nlm.nih.gov/books/NBK27118/figure/A1045/?report=objectonly
The properties of the various antigen-presenting
cells
Dendritic cells, macrophages, and B cells are the
main cell types involved in the initial
presentation of foreign antigens to naive T cells.
These cells vary in their means of antigen uptake,
MHC class II expression, co-stimulator expression,
the type of antigen they present effectively, their
locations in the body, and their surface adhesion
molecules (not shown).
http://www.ncbi.nlm.nih.gov/books/NBK27118/figure/A1046/?report=objectonly
http://www.cnic.es/en/inflamacion/proteomica/
Struttura schematica delle tetraspanine
Levy S, Shoham T. The tetraspanin web modulates immune-signalling complexes. Nat Rev
Immunol. 2005 Feb;5(2):136-48.
http://www.nature.com/nri/journal/v5/n2/fig_tab/nri1548_F1.html#figure-title
A.
E’ illustrata la CD 82 come tetraspanina tipica. I quattro
domini transmembrana ™ contengono residui polari
conservati (cerchi verdi) e affiancano gli loops
extracellulari piccolo (“small”) e grande (“large”) (SEL e
LEL, rispettivamente). Il LEL è composto da una zona
centrale formata dalle eliche a, b e d, e questa struttura
centrale è conservata fra le tetraspanine. Le eliche c e d
comprendono le porzioni variabili del LEL, e sono
affiancate dal motivo CCG e ulteriori residui di cisteina
conservati (cerchi giallo). Questa regione è ripiegata come
risultato di ponti disolfuro (linee nere) formando una sorta
di fungo. Sono anche indicati siti potenziali di
palmitoilazione nei residui intracellulari conservati di
cisteina (cerchi arancione).
B.
Una rappresentazione monomerica a nastro della
struttura tridimensionale della LEL della CD81. Le eliche
conservate a, b ed e sono colorate in blu, e la regione
divergente, che nella CD81 è rappresentata dalle eliche c e
d, è colorata rosso. I ponti disolfuro sono mostrati come
linee gialle.
C.
Modello della topologia della LEL delle tetraspanine
contenente quattro 8gruppo 1), sei (gruppo 2b) o otto
(gruppo 3) residui di cisteina nei loro LELs. Rettangoli,
frecce e linee sottili corrispondono ad eliche, filamenti e
avvolgimenti, rispettivamente. I ponti disolfuro sono
rappresentati da linee nere.
Ruolo doplice del CD81 nell’immunosinapsi tra cellula
presentante l’antigene e la cellula T
.
• In cellule che presentano l’antigene (illustrate
qui come un linfocito B), la tratraspanina CD81
si associa con molecule dei MHC di classe II,
particolarmente con il sotto-insieme
contenente l’epitopo CDw78.
• Allo stesso modo gli exosomi (illustrati come
vescicole secrete colorate in blu), che sono
arricchiti in tetraspanine, sono coinvolti nella
presentazione di antigene.
• Nei linfociti T, la CD81 si associa con CD3 e
CD4; il suo effetto co-stimolatorio, e quello
delle tetraspanine in genere, è simile a quello
del CD28.
• Ci sono anche dati che la CD81 sia distribuita al
“central supramolecular activation complex”
(cSMAC).
Levy S, Shoham T. The tetraspanin web modulates immune-signalling complexes.
Nat Rev Immunol. 2005 Feb;5(2):136-48.
http://www.nature.com/nri/journal/v5/n2/fig_tab/nri1548_F3.html#figure-title
[att.ne: Didascalie scambiate delle figure]
• Per ora, sono state descritte e rimane da
determinare se esse formano anche transinterazioni. TCR: “T-cell receptor”.
Didascalia Fig. Levy
•
In un modello ipotetico, la rete di tetraspanine aggrega transitoriamente component delle vie di segnalazione transmembrane
ed intracellulari, in questo modo facilitando una risposta specifica e altamente regolata a diversi segnali extracellulari.
• Nei leucociti circolanti, le integrine mediano in modo esclusivo la risposta cellulare sia a segnali adesivi che stimolatori
provenienti dalle cellule endoteliali, quali le chemochine. Le chemochine si legano ai loro rispettivi recettori associati a
proteine G (GPCRs) presenti sulla superficie dei leucociti, aumentano l’avidità delle integrine e aumentano l’adesività.
• Le tetraspanine si associano con le integrine e potrebbero mediare il trasferimento del segnale fra stimoli associati al GPCR
ealle integrine. In effetti, asssociazioni altamente specifiche fra le tetraspanine e membri della famiglia GPCR furono
recentemente descritti.
• Un ulteriore contributo delle tetraspanine è la loro capacità di reclutare particolari enzimi coinvolti nel segnalamento cellulare,
incluso la proteina chinasi C (PKC) che si sa essere coinvolta nella fosforilazione delle integrine, e i componenti intracellulari del
complesso GPCR, le subunità della proteina G. Inoltre, la CD81 si associa con un’isoforma che appartiene alla famiglia di
proteine 14-3-3; questa associazione dipende dallo stato di ossidazione della cellula e dalla palmitoilazione del CD81. La
famiglia 14-3-3 è stata implicata nella regolazione delle vie di segnalamento intracellulari.
• Vi sono inoltre ulteriori associazioni laterali tra le tetraspanine e altri membri della superfamiglia delle immunoglobuline, quali
EW12 (un componente della superfamiglia delle immunoglobuline che contiene un motivo EWI di aminoacidi).
• Complessivamente, la capacità delle tetraspanine di simultaneamente associarsi una con l’altra, con partners e con diverse
classi di proteine di segnalamento permette la trasmissione di segnali laterali e facilita il coordinamento degli eventi di
segnalamento intracellulare. Gα: subunità α della proteina G.
Levy S, Shoham T. The tetraspanin web modulates immune-signalling complexes. Nat Rev Immunol. 2005 Feb;5(2):136-48.
Levy S, Shoham T. Protein-protein interactions in the tetraspanin web. Physiology (Bethesda). 2005 Aug;20:218-24.
Vescicole extracellulari
FEGATO
Exosome release
(A) Exosomes containing membrane and cytosolic proteins, mRNAs, and miRNAs, are derived from the multivesicular body (MVB)
sorting pathway. Membrane proteins are oriented in a fashion (extracellular region out) that permits profound biological autocrine
and paracrine effects. (B) Exosomes isolated from rat bile have a cup- or “deflated football”- shaped morphology by transmission
electron microscopy (TEM), but they have a perfectly round shape by scanning electron microscopy (SEM). (C) In cholangiocytes of
mouse liver, MVBs containing exosomes (arrows) (a) move to the apical plasma membrane (APM) (b), and release exosomes into the
bile duct lumen by exocytosis (c).
Masyuk AI, Masyuk TV, Larusso NF. Exosomes in the pathogenesis, diagnostics and therapeutics of liver diseases. J Hepatol. 2013 Sep;59(3):621-5.
Exosomes in intercellular signaling
(A) In the liver, exosomes derived from hepatocytes and cholangiocytes are transported by
bile flow to target cholangiocytes with which they may interact via several mechanisms
depending on their cargo and biological properties. They can fuse with the plasma
membrane and deliver their content into the cytoplasm of a target cell; interact with
receptors on the apical plasma and ciliary membrane inducing intracellular signaling; and
endocytosed for recycling. (B and C) Biliary exosomes surround and attach to cholangiocyte
cilia in mouse liver as viewed by TEM (B) and SEM (C), supporting the involvement of
exosomes and cilia in mechanisms of intercellular signaling.
Masyuk AI, Masyuk TV, Larusso NF. Exosomes in the
pathogenesis, diagnostics and therapeutics of liver
diseases. J Hepatol. 2013 Sep;59(3):621-5.
Larusso NF, Masyuk TV. The role of cilia in the regulation of bile flow. Dig Dis. 2011;29(1):6-12.
Gli exosomi sono coinvolti nella funzione
chemosensoriale delle cilia primarie dei
colangiociti (cellule dei dotti biliari). Gli
exosomi sono piccole (30 -100 nm di
diametro) vescicole extracellulari rivestite da
membrana. Sono derivati da vescicole
interne di corpi multivescicolari (MVBs) che
si fondono con la membrana plasmatica in
una modalità simile all’esocitosi e rilasciano
il loro contenuto nello spazio extracellulare
(schema). La presenza di vescicole tipo
exosomi di 50-80 nm di diametro nel lume
dei dotti intraepatici di topi «wild-type» e
policistici è stata confermata da microscopia
elettronica a trasmissione (destra, panelli di
sopra). Queste vescicole circondono cilia dei
colangiociti ed alcune sembrano attaccarsi
alla membrana ciliare e dei microvilli.
L’immagine del microscopio elettronico a
scansione (SEM) (destra, panello di sotto)
suggerisce che vescicole simili ad exosomi di
fatto si leghino alle cilia.
Exosome-like vesicles surround and
attach to mouse cholangiocyte
[primary] cilia in vivo.
By transmission (TEM; A and B) and
scanning (SEM; C and D) electron
microscopy, exosome-like vesicles (black
and white arrows) are present in the
lumen of intrahepatic bile ducts in the
wild-type (A and C) and Pkhd1del2/del2
(B and D) mice. The vesicles surround
the cilium (B) and attach to this
organelle (A–D) and microvilli (A) of the
cholangiocyte apical plasma membrane.
Masyuk AI, Huang BQ, Ward CJ, Gradilone SA, Banales JM, Masyuk TV, Radtke
B, Splinter PL, LaRusso NF. Biliary exosomes influence cholangiocyte regulatory
mechanisms and proliferation through interaction with primary cilia. Am J
Physiol Gastrointest Liver Physiol. 2010 Oct;299(4):G990-9.
Hepatocyte multivesicular bodies (MVBs) and luminal vesicles are positive for an exosomal marker, CD63
[tetraspanin].
MVBs and intraluminal vesicles positive for an exosomal marker, CD63, (black arrows) were observed in normal rat
hepatocytes. MVBs are seen in a proximity to the hepatic canaliculus (a). CD63-positive vesicles are also seen in the
canalicular lumen (b), suggesting that hepatocytes release exosomes in vivo.
Masyuk AI, Huang BQ, Ward CJ, Gradilone SA, Banales JM, Masyuk TV, Radtke B, Splinter PL, LaRusso NF. Biliary exosomes influence cholangiocyte regulatory mechanisms and
proliferation through interaction with primary cilia. Am J Physiol Gastrointest Liver Physiol. 2010 Oct;299(4):G990-9.
Vescicole extracellulari
Tumori
Heterotypic cellular interactions in the tumour
microenvironment.
The tumour microenvironment is a complex scaffold of
an extracellular matrix (ECM) and various cell types. In
addition to malignant cells, vascular cells, stromal cells
and immune cells are common cellular residents of the
tumour niche. Tumour cells mould this environment
for their own needs via intercellular communication
pathways, such as direct cell-to-cell contacts and the
release of growth factors, matrix metalloproteases,
ECM proteins and extracellular vesicles (EVs). Tumour
cell-mediated stromal modifications include:
suppression of anti-tumoural immune responses,
deposition and degradation of ECM components,
induction of vascular network formation and
recruitment of stromal cells and tumour-promoting
immune cells. In turn, heterogeneous tumour
microenvironmental components create a favourable
environment for tumour growth and dissemination.
Various tumour microenvironmental stressors are
inherent features of solid tumours that profoundly
modify the tumour milieu and accelerate tumour
progression towards malignancy.
Kucharzewska P, Belting M. Emerging roles of extracellular vesicles in the adaptive response of tumour cells to microenvironmental stress. J Extracell Vesicles. 2013 Mar 5;2. doi:
10.3402/jev.v2i0.20304. eCollection 2013.
Extracellular vesicles (EVs) are potential
conveyors of stress-mediated tumour
progression.
EVs are shed from various cellular components
of the tumour milieu to mediate exchange of
signalling proteins and genetic material, which
altogether may support tumour growth and
progression. Diverse tumour
microenvironmental stress conditions augment
tumour-promoting activities of EVs by
modulating their secretion and trafficking in
the extracellular space, as well as altering their
molecular content and functional activity.
Upon release, EVs may also enter the
circulation and mediate long-range exchange of
EV-associated cargo that may support the
process of pre-metastatic niche formation. In
addition, circulating EVs carrying multifaceted,
molecular stress signatures may offer unique,
non-invasive biomarkers that can be used in
the management of cancer patients.
Kucharzewska P, Belting M. Emerging roles of extracellular
vesicles in the adaptive response of tumour cells to
microenvironmental stress. J Extracell Vesicles. 2013 Mar 5;2.
doi: 10.3402/jev.v2i0.20304. eCollection 2013.
D'Asti E, Garnier D, Lee TH, Montermini L, Meehan
B, Rak J. Oncogenic extracellular vesicles in brain
tumor progression. Front Physiol. 2012 Jul
24;3:294.
RUOLO DELLE VESCICOLE
EXTRACELLULARI NELLA REGOLAZIONE
DELL’IMMUNITA’ DEI TUMORI E DEI
MICROORGANISMI CHE PUO’ ESSERE
MODIFICATA PER APPLICAZIONI
TERAPEUTICHE
Robbins PD, Morelli AE. Regulation of immune responses by
extracellular vesicles. Nat Rev Immunol. 2014 Mar;14(3):195-208.
Contenuto e funzioni di vescicole extracellulari rilasciate
da differenti tipi di cellule tumorali – 1
Camussi G, Deregibus MC, Tetta C. Tumor-derived microvesicles and the cancer microenvironment. Curr Mol Med. 2013 Jan;13(1):58-67.
Contenuto e funzioni di vescicole extracellulari rilasciate
da differenti tipi di cellule tumorali – 2
Camussi G, Deregibus MC, Tetta C. Tumor-derived microvesicles and the cancer microenvironment. Curr Mol Med. 2013 Jan;13(1):58-67.
Contenuto e funzioni di vescicole extracellulari rilasciate
da differenti tipi di cellule tumorali – 3
Camussi G, Deregibus MC, Tetta C. Tumor-derived microvesicles and the cancer microenvironment. Curr Mol Med. 2013 Jan;13(1):58-67.
Vescicole extracellulari
Intestino
Intestinal epithelial cells (IEC)
secrete exosomes. IEC express
accessory molecules (MHC class
II, invariant chain, HLA-DM) and
are considered as nonprofessional antigen presenting
cells. The lack of direct contact
between IEC and CD4+ T cells
limits direct antigen presentation
in vivo. However, IEC secrete
exosomes which are small
membrane vesicles originating
from the MHC class II-enriched
compartment (MIIC) and are
released by exocytosis of these
compartments in the external
medium. Such epithelial
exosomes bear class II/peptide
complexes and molecules
potentially involved in cell– cell
or cell –matrix interactions.
Mallegol J, van Niel G, Heyman M. Phenotypic and functional characterization of intestinal epithelial exosomes. Blood Cells Mol Dis. 2005 Jul-Aug;35(1):11-6.
A model for the molecular structure of epithelial-derived
exosomes. Ubiquitously expressed molecules such as
enzymes of the intracellular metabolism (pyruvate kinase
M2, creatine kinase, a-enolase, phosphoglycerate kinase,
glyceraldehyde-3-phosphate dehydrogenase, L-lactate
dehydrogenase) and cytoskeleton proteins (actin,
tubulin), as well as molecules possibly involved in antigen
presentation (MHC class I, MHC class II, CD63), were
found in both apical and basolateral exosomes. Apical
exosomes also carried molecules involved in apical
addressing of endosomes (syntaxin 3, syntaxin-binding
protein 2), whereas basolateral exosomes had molecules
that might act as adhesion or costimulatory molecules
(A33 antigen and epithelial cell surface antigen).
van Niel G, Raposo G, Candalh C, Boussac M, Hershberg R, Cerf-Bensussan N, Heyman M. Intestinal epithelial cells secrete exosome-like vesicles. Gastroenterology. 2001
Aug;121(2):337-49.
INTESTINO
VIE DI TRASPORTO PARACELLULARE
Under steady-state condition, molecules of molecular weight
(MW) > 600 Da (such as food antigens, peptides) are
sampled by the epithelial cells by endocytosis at the apical
membrane and transcytosis toward the lamina propria.
During transcytosis, full-length peptides or proteins are
partly degraded in acidic and lysosomal compartments and
released in the form of amino acids (total degradation) or
breakdown products (partial degradation) at the basolateral
pole of enterocytes. Early endosomes containing partially
degraded food antigens meet the major histocompatibility
complex (MHC) class II-enriched compartment (MIIC) where
exogenous peptides are loaded on MHC class II molecules.
Inward invagination of MIIC compartment lead to the
formation of exosomes, which are small membrane vesicles
(40 – 90 nm) bearing MHC class II / peptide complexes at
their surface. Exosomes can diffuse in the basement
membrane and interact with local immune cells. Exosomebound peptides are much more potent than free peptides to
interact with dendritic cells and stimulate peptide
presentation to T cells.
Ménard S, Cerf-Bensussan N, Heyman M. Multiple facets of intestinal permeability and epithelial handling of dietary antigens. Mucosal Immunol. 2010 May;3(3):247-59.
Vescicole extracellulari
Cervello
Sáenz-Cuesta M, Osorio-Querejeta I, Otaegui D. Extracellular Vesicles in Multiple Sclerosis: What are They Telling Us? Front Cell Neurosci. 2014 Mar 28;8:100. doi:
10.3389/fncel.2014.00100. eCollection 2014.
Extracellular membrane vesiclesmediated mechanisms in neurons.
(A) A gradient of EMVs in the
developing nervous system can serve as
a directional guide to axonal growth.
(B) EMVs released from presynaptic
nerve terminals and taken up by their
postsynaptic partners can carry
informational content which can
modulate the strength of synaptic
activity.
(C) Regeneration of peripheral nerves is
enhanced by the EMV transfer of
ribosomes and mRNA directly from
surrounding Schwann cells into the
injured nerve to promote protein
synthesis.
Lai CP, Breakefield XO. Role of exosomes/microvesicles in the nervous system and use in emerging therapies. Front Physiol. 2012 Jun 27;3:228. doi:
10.3389/fphys.2012.00228. eCollection 2012.
Vesicle-mediated signaling at the nodes of
Ranvier. At the node, the axon is no longer
closely surrounded by compact myelin as at
the internodes (not shown), but
is covered by microvilli and rings of the
Schwann cell. This is the site at which nonsecretory vesicles of the Schwann cells
(blue) are exocytized, inducing a local
increase of
the surface area probably necessary for the
budding of shedding vesicles (red). The
shedding vesicles can either fuse with the
plasma membrane of the axon or be taken
up
by endocytosis. Both the direct fusion of the
vesicle membrane with the plasma
membrane and their membrane fusion with
the endosomal membrane release the cargo
containing RNAs, lipids and proteins. The
arrows indicate the direction of the
intracellular traffic of shedding vesicles; the
arrowheads indicate the direction of the
cargo
discharge from fused shedding vesicles.
Cocucci E, Racchetti G, Meldolesi J. Shedding microvesicles: artefacts no more. Trends Cell Biol. 2009 Feb;19(2):43-51.
Lai CP, Breakefield XO. Role of exosomes/microvesicles in the nervous system and use in emerging therapies. Front Physiol. 2012 Jun 27;3:228. doi:
10.3389/fphys.2012.00228. eCollection 2012.
Extracellular membrane vesicles-based
therapies.
(A) EMV immunotherapy. EMVs containing
tumor-antigen within and/or on the
membrane surface are isolated from
different sources and introduced in vivo to
elicit targeted immuneresponses.
(B) EMV RNAi therapy. EMVs derived from
immature dendritic cells (DCs) expressing
Rabies glycoprotein-Lamp2b fusion protein
were electroporated with siRNAs for
targeting against neurons, microglia, and
oligodendrocytes for subsequent gene
silencing.
(C) EMV drug therapy. Therapeutic
compounds can be packaged into/onto
EMVs isolated from donor cells to minimize
degradation and increase delivery to
intended sites.
EMV: extracellular membrane vesicles
Sáenz-Cuesta M, Osorio-Querejeta I, Otaegui D. Extracellular Vesicles in Multiple Sclerosis: What are They Telling Us? Front Cell Neurosci. 2014 Mar 28;8:100. doi:
10.3389/fncel.2014.00100. eCollection 2014.
Vescicole extracellulari
Cellule staminali
Camussi G, Deregibus MC, Bruno S, Cantaluppi V, Biancone L. Exosomes/microvesicles as a mechanism of cell-to-cell communication. Kidney Int.
78: 838-848, 2010.
TOLEROSOMES
Admyre C, Telemo E, Almqvist N, Lötvall J, Lahesmaa R, Scheynius A, Gabrielsson S. Exosomes - nanovesicles with possible roles in allergic inflammation. Allergy. 2008
Apr;63(4):404-8.
Extracellular vesicles contribute to
inflammation.
Recognition of PAMPs and DAMPS by APC can
cause inflammation via the secretion of
proinflammatory cytokines such as IL-1β. a |
Pathogens secrete EVs that carry PAMPs and trigger
inflammation. Different types of cell death result in
release of EVs that contain DAMPs. EVs from
pyroptotic cells have leaky membranes and release
endogenous danger signals. Apoptotic vesicles
contain high concentrations of the alarmin,
HMGB1. b | Autoantigens in EVs (yellow circles) are
recognized by autoantibodies and form immune
complexes. c | Cytokines are associated with EVs.
IL-1β is released by cells as a cleaved active
cytokine, secreted by microvesicles from the cell
surface, or is released via exosomes. Activation of
the P2X purinoreceptor-7 by ATP causes IL-1β
release from the vesicle lumen into the
extracellular space. Abbreviations: APC, antigen
presenting cell; DAMPs, damage-associated
molecular patterns; EVs, extracellular vesicles;
HMGB1, high mobility group Box 1; HSP, heat-shock
protein; NLR, NOD-like receptor; PAMPs, pathogenassociated molecular patterns; TLR, Toll-like
receptor.
Buzas EI, György B, Nagy G, Falus A, Gay S. Emerging role of extracellular vesicles in inflammatory diseases. Nat Rev
Rheumatol. 2014 Feb 18. doi: 10.1038/nrrheum.2014.19. [Epub ahead of print]
Prof.Dr. Hans-Hermann Gerdes
Scanning electron micrograph showing a thin
membrane tube (referred to as tunneling
nanotube, TNT) connecting two cultured PC12
cells. Bar, 10 mm.
Tunneling nanotubes, a new route for cell-to-cell
communication
Recently, we have discovered that cells are connected by thin
membrane tubes … These tubes, referred to as tunneling
nanotubes (TNTs), mediate membrane continuity between
connected cells and lead to complex cellular networks. TNTs
were shown to accomplish the selective uni-directional transfer
of endosome-like organelles as well as, on a limited scale, of
membrane components and cytoplasmic molecules. The data
suggest a new biological principle of cell-to-cell
communication based on membrane continuity and
intercellular transfer of cellular components like organelles
and signaling molecules. It now emerges that TNT-based
communication is a widespread mechanism throughout the
animal kingdom which includes cellular differentiation,
proliferation and development of diseases … . Based on these
findings, our research is focusing on the characterization of the
structure and function of TNTs in various cell systems. This
includes primary cultures of astrocytes and neurons.
http://www.uni-heidelberg.de/izn/researchgroups/gerdes/currentresearch.html
Trogocitosi
In the immune system many ways of cell-to-cell
communication are described, one of which is
trogocytosis (from Greek trogo-, nibble).
Trogocytosis is a poorly defined process by
which intact proteins, protein-complexes and
even membrane patches are transferred from
one cell to another.
Here we review the literature focusing on an
article of Martínez-Martín et al. (Immunity,
2011) who experimentally showed that the
central supramolecular activation cluster
(cSMAC) of the immune-synapse, functions as a
site of clathrin-independent T cell receptor (TCR)
internalization and trogocytosis. For the first
time, the paper uncovers molecular players
involved in the process of trogocytosis.
Surprisingly, these share key features of
phagocytosis. Further, they identify small Rho
GTPases TC21 and RhoG as key mediators of
these processes.
Dopfer EP, Minguet S, Schamel WW. A new vampire saga: the molecular mechanism of T cell trogocytosis. Immunity. 2011 Aug 26;35(2):151-3
VEXOSOMI
Overview of adeno-associated virus (AAV) vexosome vector system. (a) AAV is conventionally purified
from cell lysates (i), However, AAV is also shed into the media apparently as both free particles32 (ii), as
well as microvesicle-associated vectors (vexosomes) (iii). Vexosomes can be isolated from the media and
used for gene delivery to target cells. (b) Illustration of vexosome components. The microvesicle may
contain AAV vectors encoding a gene of interest, mRNA, miRNA or DNA as well as vector encoded and
nonvector-encoded proteins (orange). The microvesicle surface may be decorated with receptors or
ligands endogenously (yellow) or ectopically (green) expressed in the packaging cell.
Maguire CA, Balaj L, Sivaraman S, Crommentuijn MH, Ericsson M, Mincheva-Nilsson L, Baranov V, Gianni D, Tannous BA, Sena-Esteves M, Breakefield XO, Skog J. Microvesicleassociated AAV vector as a novel gene delivery system. Mol Ther. 2012 May;20(5):960-71.

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