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Exosomi Risposta immunitaria SEMINARIO. Théry C, Ostrowski M, Segura E. Membrane vesicles as conveyors of immune responses. Nat Rev Immunol. 2009 Aug;9(8):581-93.

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Presentazione sul tema: "Exosomi Risposta immunitaria SEMINARIO. Théry C, Ostrowski M, Segura E. Membrane vesicles as conveyors of immune responses. Nat Rev Immunol. 2009 Aug;9(8):581-93."— Transcript della presentazione:

1 Exosomi Risposta immunitaria SEMINARIO

2 Théry C, Ostrowski M, Segura E. Membrane vesicles as conveyors of immune responses. Nat Rev Immunol Aug;9(8): 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.

3 Théry C, Ostrowski M, Segura E. Membrane vesicles as conveyors of immune responses. Nat Rev Immunol Aug;9(8): 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 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.

4 Davis DM. Intercellular transfer of cell-surface proteins is common and can affect many stages of an immune response. Nat Rev Immunol Mar;7(3): Meccanismi per il trasferimento intercellulare di proteine di membrana tra cellule immunitarie (Fig.1)

5 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 Mar;7(3):

6 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 Mar;7(3):

7 Mittelbrunn M, Sánchez-Madrid F. Intercellular communication: diverse structures for exchange of genetic information. Nat Rev Mol Cell Biol Apr 18;13(5): 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 “peptide- loaded 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.

8 Processi in cui sono coinvolte microvescicole Presentatione di antigene

9 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.

10 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 co- stimolatorie 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”.

11 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 antigen- presenting 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.

12 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 antigen- specific 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.

13 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.

14 The principal co-stimulatory molecules expressed on antigen- presenting 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.

15 The requirement for one cell to deliver both the antigen- specific 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.

16 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. Presentazione di antigene da parte delle cellule dendritiche

17 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 co- stimulatory B7 molecules in the antigen-presenting cells. Such added bacteria act as adjuvants.

18 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).

19 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).

20

21 Levy S, Shoham T. The tetraspanin web modulates immune-signalling complexes. Nat Rev Immunol Feb;5(2): Struttura schematica delle tetraspanine 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.

22 . Levy S, Shoham T. The tetraspanin web modulates immune-signalling complexes. Nat Rev Immunol Feb;5(2): [att.ne: Didascalie scambiate delle figure] 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). Per ora, sono state descritte e rimane da determinare se esse formano anche trans- interazioni. TCR: “T-cell receptor”.

23 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 ; questa associazione dipende dallo stato di ossidazione della cellula e dalla palmitoilazione del CD81. La famiglia 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 Feb;5(2):

24 Levy S, Shoham T. Protein-protein interactions in the tetraspanin web. Physiology (Bethesda) Aug;20:

25 Vescicole extracellulari FEGATO

26 Masyuk AI, Masyuk TV, Larusso NF. Exosomes in the pathogenesis, diagnostics and therapeutics of liver diseases. J Hepatol Sep;59(3): 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).

27 Masyuk AI, Masyuk TV, Larusso NF. Exosomes in the pathogenesis, diagnostics and therapeutics of liver diseases. J Hepatol Sep;59(3): 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.

28 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 ( 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 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.

29 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 Oct;299(4):G 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.

30 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 Oct;299(4):G 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.

31 Vescicole extracellulari Tumori

32 Kucharzewska P, Belting M. Emerging roles of extracellular vesicles in the adaptive response of tumour cells to microenvironmental stress. J Extracell Vesicles Mar 5;2. doi: /jev.v2i eCollection 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.

33 Kucharzewska P, Belting M. Emerging roles of extracellular vesicles in the adaptive response of tumour cells to microenvironmental stress. J Extracell Vesicles Mar 5;2. doi: /jev.v2i eCollection 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.

34 D'Asti E, Garnier D, Lee TH, Montermini L, Meehan B, Rak J. Oncogenic extracellular vesicles in brain tumor progression. Front Physiol Jul 24;3:294.

35 Robbins PD, Morelli AE. Regulation of immune responses by extracellular vesicles. Nat Rev Immunol Mar;14(3): RUOLO DELLE VESCICOLE EXTRACELLULARI NELLA REGOLAZIONE DELL’IMMUNITA’ DEI TUMORI E DEI MICROORGANISMI CHE PUO’ ESSERE MODIFICATA PER APPLICAZIONI TERAPEUTICHE

36 Camussi G, Deregibus MC, Tetta C. Tumor-derived microvesicles and the cancer microenvironment. Curr Mol Med Jan;13(1): Contenuto e funzioni di vescicole extracellulari rilasciate da differenti tipi di cellule tumorali – 1

37 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 Jan;13(1):58-67.

38 Contenuto e funzioni di vescicole extracellulari rilasciate da differenti tipi di cellule tumorali – 3

39 Vescicole extracellulari Intestino

40 Mallegol J, van Niel G, Heyman M. Phenotypic and functional characterization of intestinal epithelial exosomes. Blood Cells Mol Dis Jul-Aug;35(1):11-6. Intestinal epithelial cells (IEC) secrete exosomes. IEC express accessory molecules (MHC class II, invariant chain, HLA-DM) and are considered as non- professional 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.

41 van Niel G, Raposo G, Candalh C, Boussac M, Hershberg R, Cerf-Bensussan N, Heyman M. Intestinal epithelial cells secrete exosome-like vesicles. Gastroenterology Aug;121(2): 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).

42 Ménard S, Cerf-Bensussan N, Heyman M. Multiple facets of intestinal permeability and epithelial handling of dietary antigens. Mucosal Immunol May;3(3): 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. Exosome- bound peptides are much more potent than free peptides to interact with dendritic cells and stimulate peptide presentation to T cells. INTESTINO VIE DI TRASPORTO PARACELLULARE

43 Vescicole extracellulari Cervello

44 Sáenz-Cuesta M, Osorio-Querejeta I, Otaegui D. Extracellular Vesicles in Multiple Sclerosis: What are They Telling Us? Front Cell Neurosci Mar 28;8:100. doi: /fncel eCollection 2014.

45 Lai CP, Breakefield XO. Role of exosomes/microvesicles in the nervous system and use in emerging therapies. Front Physiol Jun 27;3:228. doi: /fphys eCollection Extracellular membrane vesicles- mediated 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.

46 Cocucci E, Racchetti G, Meldolesi J. Shedding microvesicles: artefacts no more. Trends Cell Biol Feb;19(2): 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 non- secretory 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.

47 Lai CP, Breakefield XO. Role of exosomes/microvesicles in the nervous system and use in emerging therapies. Front Physiol Jun 27;3:228. doi: /fphys eCollection 2012.

48 Sáenz-Cuesta M, Osorio-Querejeta I, Otaegui D. Extracellular Vesicles in Multiple Sclerosis: What are They Telling Us? Front Cell Neurosci Mar 28;8:100. doi: /fncel eCollection 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

49 Vescicole extracellulari Cellule staminali

50 Camussi G, Deregibus MC, Bruno S, Cantaluppi V, Biancone L. Exosomes/microvesicles as a mechanism of cell-to-cell communication. Kidney Int. 78: , 2010.

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53 Admyre C, Telemo E, Almqvist N, Lötvall J, Lahesmaa R, Scheynius A, Gabrielsson S. Exosomes - nanovesicles with possible roles in allergic inflammation. Allergy Apr;63(4): TOLEROSOMES

54 Buzas EI, György B, Nagy G, Falus A, Gay S. Emerging role of extracellular vesicles in inflammatory diseases. Nat Rev Rheumatol Feb 18. doi: /nrrheum [Epub ahead of print] 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, pathogen- associated molecular patterns; TLR, Toll-like receptor.

55 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. Prof.Dr. Hans-Hermann Gerdes

56 Dopfer EP, Minguet S, Schamel WW. A new vampire saga: the molecular mechanism of T cell trogocytosis. Immunity Aug 26;35(2):151-3 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.

57 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 particles 32 (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. Microvesicle- associated AAV vector as a novel gene delivery system. Mol Ther May;20(5): VEXOSOMI


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