Induction of Lymphocyte Apoptosis by Tumor Cell Secretion of FasL-bearing Microvesicles

The hypothesis that FasL expression by tumor cells may impair the in vivo efficacy of antitumor immune responses, through a mechanism known as ‘Fas tumor counterattack,’ has been recently questioned, becoming the object of an intense debate based on conflicting results. Here we definitely show that FasL is indeed detectable in the cytoplasm of melanoma cells and its expression is confined to multivesicular bodies that contain melanosomes. In these structures FasL colocalizes with both melanosomal (i.e., gp100) and lysosomal (i.e., CD63) antigens. Isolated melanosomes express FasL, as detected by Western blot and cytofluorimetry, and they can exert Fas-mediated apoptosis in Jurkat cells. We additionally show that melanosome-containing multivesicular bodies degranulate extracellularly and release FasL-bearing microvesicles, that coexpress both gp100 and CD63 and retain their functional activity in triggering Fas-dependent apoptosis of lymphoid cells. Hence our data provide evidence for a novel mechanism potentially operating in Fas tumor counterattack through the secretion of subcellular particles expressing functional FasL. Such vesicles may form a sort of front line hindering lymphocytes and other immunocompetent cells from entering neoplastic lesions and exert their antitumor activity

Natural Killer and NK-Like T-Cell Activation in Colorectal Carcinoma Patients Treated with Autologous Tumor-Derived Heat Shock Protein 96

Heat shock proteins (HSPs) are involved in the activation of both adaptive and innate immune systems. Here, we report that vaccination with autologous tumor-derived HSP96 of colorectal cancer patients, radically resected for liver metastases, induced a significant boost of natural killer (NK) activity detected as cytokine secretion and cytotoxicity in the presence of NK-sensitive targets. Increased NK activity was associated with a raise in CD3−CD56+ NK and/or CD3+CD56+ NK-like T cells, displaying enhanced expression of NKG2D and/or NKp46 receptors. Up-regulated expression of CD83 and CD40 and increased interleukin-12 release on stimulation were observed in CD14+ cells from post-HSP96 peripheral blood mononuclear cells, suggesting an indirect pathway of NK stimulation by HSP96-activated monocytes. Additionally, CD3−CD56+ and CD3+CD56+ lymphocytes were found to undergo functional and phenotypic activation on in vitro exposure to HSP96 even in the absence of monocytes, supporting a potential direct activity of HSP96 on these cell subsets. This evidence was confirmed by the specific binding of FITC-conjugated HSP96 to a subset of both CD3−CD56+ and CD3+CD56+ cells in peripheral blood mononuclear cells from colorectal cancer patients. Altogether, these findings identify the activation of the NK compartment as an additional immunologic effect of autologous tumor-derived HSP96 administration in cancer patients

Human Tumor-Derived Heat Shock Protein 96 Mediates In Vitro Activation and In Vivo Expansion of Melanoma- and Colon Carcinoma-Specific T Cells

Abstract Heat shock proteins (hsp) 96 play an essential role in protein metabolism and exert stimulatory activities on innate and adaptive immunity. Vaccination with tumor-derived hsp96 induces CD8+ T cell-mediated tumor regressions in different animal models. In this study, we show that hsp96 purified from human melanoma or colon carcinoma activate tumor- and Ag-specific T cells in vitro and expand them in vivo. HLA-A*0201-restricted CD8+ T cells recognizing Ags expressed in human melanoma (melanoma Ag recognized by T cell-1 (MART-1)/melanoma Ag A (Melan-A)) or colon carcinoma (carcinoembryonic Ag (CEA)/epithelial cell adhesion molecule (EpCAM)) were triggered to release IFN-γ and to mediate cytotoxic activity by HLA-A*0201-matched APCs pulsed with hsp96 purified from tumor cells expressing the relevant Ag. Such activation occurred in class I HLA-restricted fashion and appeared to be significantly higher than that achieved by direct peptide loading. Immunization with autologous tumor-derived hsp96 induced a significant increase in the recognition of MART-1/Melan-A27–35 in three of five HLA-A*0201 melanoma patients, and of CEA571–579 and EpCAM263–271 in two of five HLA-A*0201 colon carcinoma patients, respectively, as detected by ELISPOT and HLA/tetramer staining. These increments in Ag-specific T cell responses were associated with a favorable disease course after hsp96 vaccination. Altogether, these data provide evidence that hsp96 derived from human tumors can present antigenic peptides to CD8+ T cells and activate them both in vitro and in vivo, thus representing an important tool for vaccination in cancer patients

Il Futuro della Cybersecurity in Italia: Ambiti Progettuali Strategici

Il presente volume nasce come continuazione del precedente, con l’obiettivo di delineare un insieme di ambiti progettuali e di azioni che la comunità nazionale della ricerca ritiene essenziali a complemento e a supporto di quelli previsti nel DPCM Gentiloni in materia di sicurezza cibernetica, pubblicato nel febbraio del 2017. La lettura non richiede particolari conoscenze tecniche; il testo è fruibile da chiunque utilizzi strumenti informatici o navighi in rete. Nel volume vengono considerati molteplici aspetti della cybersecurity, che vanno dalla definizione di infrastrutture e centri necessari a organizzare la difesa alle azioni e alle tecnologie da sviluppare per essere protetti al meglio, dall’individuazione delle principali tecnologie da difendere alla proposta di un insieme di azioni orizzontali per la formazione, la sensibilizzazione e la gestione dei rischi. Gli ambiti progettuali e le azioni, che noi speriamo possano svilupparsi nei prossimi anni in Italia, sono poi accompagnate da una serie di raccomandazioni agli organi preposti per affrontare al meglio, e da Paese consapevole, la sfida della trasformazione digitale. Le raccomandazioni non intendono essere esaustive, ma vanno a toccare dei punti che riteniamo essenziali per una corretta implementazione di una politica di sicurezza cibernetica a livello nazionale. Politica che, per sua natura, dovrà necessariamente essere dinamica e in continua evoluzione in base ai cambiamenti tecnologici, normativi, sociali e geopolitici. All’interno del volume, sono riportati dei riquadri con sfondo violetto o grigio; i primi sono usati nel capitolo introduttivo e nelle conclusioni per mettere in evidenza alcuni concetti ritenuti importanti, i secondi sono usati negli altri capitoli per spiegare il significato di alcuni termini tecnici comunemente utilizzati dagli addetti ai lavori. In conclusione, ringraziamo tutti i colleghi che hanno contribuito a questo volume: un gruppo di oltre 120 ricercatori, provenienti da circa 40 tra Enti di Ricerca e Università, unico per numerosità ed eccellenza, che rappresenta il meglio della ricerca in Italia nel settore della cybersecurity. Un grazie speciale va a Gabriella Caramagno e ad Angela Miola che hanno contribuito a tutte le fasi di produzione del libro. Tra i ringraziamenti ci fa piacere aggiungere il supporto ottenuto dai partecipanti al progetto FILIERASICURA