Exosomes released by EBV-infected nasopharyngeal carcinoma cells convey the viral Latent Membrane Protein 1 and the immunomodulatory protein galectin 9

BACKGROUND: Nasopharyngeal carcinomas (NPC) are consistently associated with the Epstein-Barr virus (EBV). Their malignant epithelial cells contain the viral genome and express several antigenic viral proteins. However, the mechanisms of immune escape in NPCs are still poorly understood. EBV-transformed B-cells have been reported to release exosomes carrying the EBV-encoded latent membrane protein 1 (LMP1) which has T-cell inhibitory activity. Although this report suggested that NPC cells could also produce exosomes carrying immunosuppressive proteins, this hypothesis has remained so far untested. METHODS: Malignant epithelial cells derived from NPC xenografts – LMP1-positive (C15) or negative (C17) – were used to prepare conditioned culture medium. Various microparticles and vesicles released in the culture medium were collected and fractionated by differential centrifugation. Exosomes collected in the last centrifugation step were further purified by immunomagnetic capture on beads carrying antibody directed to HLA class II molecules. Purified exosomes were visualized by electron microscopy and analysed by western blotting. The T-cell inhibitory activities of recombinant LMP1 and galectin 9 were assessed on peripheral blood mononuclear cells activated by CD3/CD28 cross-linking. RESULTS: HLA-class II-positive exosomes purified from C15 and C17 cell supernatants were containing either LMP1 and galectin 9 (C15) or galectin 9 only (C17). Recombinant LMP1 induced a strong inhibition of T-cell proliferation (IC50 = 0.17 nM). In contrast recombinant galectin 9 had a weaker inhibitory effect (IC50 = 46 nM) with no synergy with LMP1. CONCLUSION: This study provides the proof of concept that NPC cells can release HLA class-II positive exosomes containing galectin 9 and/or LMP1. It confirms that the LMP1 molecule has intrinsic T-cell inhibitory activity. These findings will encourage investigations of tumor exosomes in the blood of NPC patients and assessment of their effects on various types of target cells

Combined strategy based on pre-activated analogs of oxazaphosphorines for increased therapeutic index and immune modulation

Oxazaphosphorines (Oxaza) represented by cyclophosphamide (CPA) and ifosfamide (IFO) are still the corner stone of several polychemotherapy protocols as they are widely indicated in the treatment of numerous cancer from soft tissue sarcomas to lymphomas and immune-related diseases. However, Oxaza are prodrugs requiring cytochrome (CYP) P450 bioactivation responsible of limiting adverse effects. In the case of IFO, bioactivation leads to a low release of 4-OH-IFO (10%), which generates the active nitrogen mustard displaying DNA cross-links. Associated toxicities of IFO due to acrolein, (urotoxicity) and to chloroacetaldehyde (neuro and nephrotoxicity) have been described. Thus, increasing IFO therapeutic index could be of major interest. To circumvent these toxicities, our team has designed new pre-activated IFO analogs to avoid CYP bioactivation (Skarbek et al J Med Chem 2015). Among these analogues some have the ability to self-assemble as nanoassemblies (NAs), the others can be encapsulated within nano-lipid capsules (NLCs). These new drug delivery systems (DDS) can take advantage of passive targeting, as stealthiness of these DDS can be provided by PEGylation by using Cholesterol-polyethylene glycol or the use of surfactant. These DDS can also be functionalized by appropriate monoclonal antibodies leading to multi stage DDS with active targeting properties. Regarding CPA, it has been shown and described in literature that low doses of CPA enhance the immunity by promoting differentiation of CD4⁺ cell toward Th1. As IFO is isomeric form of CPA, it was assumed that IFO could also have such properties. Studies on immunocompetent MCA205 mouse model, an immunogenic fibrosarcoma mouse model, demonstrate a dose-dependent immunomodulation of IFO towards a modulation of the secretion of IFNy, IL-17A and IL-6 cytokines. The ongoing experiments on mouse model depleted in CD4⁺ T cells and CD8⁺ T cells show the antitumor efficacy of IFO 150mg/kg on these immune cells in tumor regression. Both strategies could lead to the design of nano-immuno-conjugates (NICs) which could benefit of the immunomodulatory effects of X-Oxaza combined to their antiproliferative properties targeted through immune checkpoint antibodies. These new functionalized DDS may provide a useful strategy to give specificity to active drugs used for many years in clinical practice. Both DDS could be grafted with mAbs which could lead to a new family of DDS aiming to combine antiproliferative and immunomodulatory properties for a dual antitumoral action Citation Format: Julia Delahousse, Charles Skarbek, Valentine Gauthier, M Desbois, Emilie Roger, C. Pioche-Durieu, M. Rivard, D. Desmaële, T. Martens, E. LeCam, Jean-Pierre Benoit, P. Couvreur, Nathalie Chaput-Gras, Angelo Paci. Combined strategy based on pre-activated analogs of oxazaphosphorines for increased therapeutic index and immune modulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2195. doi:10.1158/1538-7445.AM2017-219

Tumor-Associated Macrophages (TAMs) Form an Interconnected Cellular Supportive Network in Anaplastic Thyroid Carcinoma

BACKGROUND: A relationship between the increased density of tumor-associated macrophages (TAMs) and decreased survival was recently reported in thyroid cancer patients. Among these tumors, anaplastic thyroid cancer (ATC) is one of the most aggressive solid tumors in humans. TAMs (type M2) have been recognized as promoting tumor growth. The purpose of our study was to analyze with immunohistochemistry the presence of TAMs in a series of 27 ATC. METHODOLOGY/PRINCIPAL FINDINGS: Several macrophages markers such as NADPH oxidase complex NOX2-p22phox, CD163 and CD 68 were used. Immunostainings showed that TAMs represent more than 50% of nucleated cells in all ATCs. Moreover, these markers allowed the identification of elongated thin ramified cytoplasmic extensions, bestowing a "microglia-like" appearance on these cells which we termed "Ramified TAMs" (RTAMs). In contrast, cancer cells were totally negative. Cellular stroma was highly simplified since apart from cancer cells and blood vessels, RTAMs were the only other cellular component. RTAMs were evenly distributed and intermingled with cancer cells, and were in direct contact with other RTAMs via their ramifications. Moreover, RTAMs displayed strong immunostaining for connexin Cx43. Long chains of interconnected RTAMs arose from perivascular clusters and were dispersed within the tumor parenchyma. When expressed, the glucose transporter Glut1 was found in RTAMs and blood vessels, but rarely in cancer cells. CONCLUSION: ATCs display a very dense network of interconnected RTAMs in direct contact with intermingled cancer cells. To our knowledge this is the first time that such a network is described in a malignant tumor. This network was found in all our studied cases and appeared specific to ATC, since it was not found in differentiated thyroid cancers specimens. Taken together, these results suggest that RTAMs network is directly related to the aggressiveness of the disease via metabolic and trophic functions which remain to be determined

Epstein-Barr virus: clinical and epidemiological revisits and genetic basis of oncogenesis

Epstein-Barr virus (EBV) is classified as a member in the order herpesvirales, family herpesviridae, subfamily gammaherpesvirinae and the genus lymphocytovirus. The virus is an exclusively human pathogen and thus also termed as human herpesvirus 4 (HHV4). It was the first oncogenic virus recognized and has been incriminated in the causation of tumors of both lymphatic and epithelial nature. It was reported in some previous studies that 95% of the population worldwide are serologically positive to the virus. Clinically, EBV primary infection is almost silent, persisting as a life-long asymptomatic latent infection in B cells although it may be responsible for a transient clinical syndrome called infectious mononucleosis. Following reactivation of the virus from latency due to immunocompromised status, EBV was found to be associated with several tumors. EBV linked to oncogenesis as detected in lymphoid tumors such as Burkitt's lymphoma (BL), Hodgkin's disease (HD), post-transplant lymphoproliferative disorders (PTLD) and T-cell lymphomas (e.g. Peripheral T-cell lymphomas; PTCL and Anaplastic large cell lymphomas; ALCL). It is also linked to epithelial tumors such as nasopharyngeal carcinoma (NPC), gastric carcinomas and oral hairy leukoplakia (OHL). In vitro, EBV many studies have demonstrated its ability to transform B cells into lymphoblastoid cell lines (LCLs). Despite these malignancies showing different clinical and epidemiological patterns when studied, genetic studies have suggested that these EBV- associated transformations were characterized generally by low level of virus gene expression with only the latent virus proteins (LVPs) upregulated in both tumors and LCLs. In this review, we summarize some clinical and epidemiological features of EBV- associated tumors. We also discuss how EBV latent genes may lead to oncogenesis in the different clinical malignancie

Uncoupling of the Hippo and Rho pathways allows megakaryocytes to escape the tetraploid checkpoint

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Blood diffusion and Th1-suppressive effects of galectin-9-containing exosomes released by Epstein-Barr virus-infected nasopharyngeal carcinoma cells.

Epstein-Barr virus (EBV)-associated nasopharyngeal carcinoma (NPC) is the third most frequent virus-associated human malignancy. How this tumor escapes immune recognition despite the expression of several viral antigens has remained poorly understood. Our previous in vitro studies have shown that NPC cells release exosomes containing high amounts of galectin-9, a ligand of the membrane receptor Tim-3, which is able to induce apoptosis in mature Th1 lymphocytes. Here, we sought to determine whether galectin-9-carrying exosomes were produced in NPC patients and whether such exosomes might play a role in the immune evasion of NPC cells. We report that galectin-9-containing exosomes are selectively detected in plasma samples from NPC patients and mice xenografted with NPC tumors. The incorporation into exosomes protects galectin-9 against proteolytic cleavage but retains its Tim-3-binding capacity. Importantly, NPC exosomes induce massive apoptosis in EBV-specific CD4+ cells used as a model of target T cells. This effect is inhibited by both anti-Tim-3 and anti-galectin-9 blocking antibodies. These results indicate that blocking galectin-9/Tim-3 interaction in vivo might alleviate the Th1 suppressive effect of NPC exosomes and sustain anti-tumoral T cell responses, and thereby improve clinical efficacy of immunotherapeutic approaches against NPC

Immunophenotypical and functional heterogeneity of dendritic cells generated from murine bone marrow cultured with different cytokine combinations: implications for anti-tumoral cell therapy

Dendritic cells (DC) are professional antigen-presenting cells that can be used as immune adjuvant for anti-tumoural therapies. This approach requires the generation of large quantities of DC that are fully characterized on the immunophenotypical and functional levels. In a murine model, we analysed the in vitro effects of granulocyte–macrophage colony-stimulating factor (GM-CSF) alone or combined with interleukin-4 (IL-4) or Flt3 ligand (Flt3-L) on the number, immunophenotype and functions of bone marrow-derived DC. In GM-CSF cultures, we have identified two populations based on their level of expression of major histocompatibility complex (MHC) class II molecules: MHC-IIhi cells, exhibiting the typical morphology and immunophenotype of myeloid DC (CD11c+ 33D1+ DEC-205+ F4/80+), and MHC-IIlo cells, heterogeneous for DC markers (30% CD11c+; 50% 33D1+; DEC-205−; F4/80+). The addition of Flt3-L to GM-CSF induced a twofold increase in MHC-IIhi DC number; besides, the MHC-IIlo cells lost all DC markers. In contrast, after addition of IL-4 to GM-CSF, the two populations displayed a very similar phenotype (CD11c+ 33D1− DEC-205+ F4/80−), differing only in their expression levels of MHC class II and costimulatory molecules, and showed similar stimulatory activity in mixed leucocyte reaction. We next analysed the migration of these cultured cells after fluorescent labelling. Twenty-four hours after injection into the footpads of mice, fluorescent cells were detected in the draining popliteal lymph nodes, with an enhanced migration when cells were cultured with GM-CSF+Flt3-L. Finally, we showed that MHC-IIhi were more efficient than MHC-IIlo cells in an anti-tumoral vaccination protocol. Altogether, our data highlight the importance of characterizing in vitro-generated DC before use in immunotherapy

Cellular but not humoral immune responses generated by vaccination with dendritic cells protect mice against leukaemia

Dendritic cells (DC) are extremely efficient at generating both prophylactic and therapeutic anti‐tumour immunity. We aimed to analyse the respective roles of humoral and cellular immune responses generated in mice vaccinated with bone marrow (BM)‐derived DC in terms of in vivo anti‐leukaemia effect. We used the murine L1210 B lymphocytic leukaemia genetically modified to express on the cell surface of human CD4 (hCD4) (L1210/hCD4) as a model tumour‐associated antigen (TAA). DC cultures were loaded with either purified soluble hCD4 (shCD4) protein or unfractionated L1210/hCD4 extracts and injected as vaccine into mice. The efficacy of these vaccinations was compared with that of vaccination with shCD4 protein emulsified in Freund’s adjuvant (FA). We evaluated the immune responses generated after these vaccinal protocols and the survival rate of vaccinated mice subsequently challenged with a lethal injection of L1210/hCD4 cells. Our results demonstrated that vaccination with shCD4 protein or tumour extract‐loaded DC mainly generated an hCD4 antigen‐specific cell‐mediated cytotoxic immune response that was associated with a specific protection against leukaemia. In contrast, vaccination with the protein emulsified in FA only generated potent humoral immune responses that were not protective against leukaemia. Altogether, our results indicate that the unique property of loaded DC to trigger an anti‐leukaemia protective effect is mainly associated with cellular immune responses