<p>PDF file - 214K, Supplemental Figure 6: Monocyte-derived DC exhibit a similar level of NYESO-1 cross-presentation compared to pDC. (A) on day0, pDC and monocytes from a single donor were purified. pDC were frozen and monocytes cultured with IL-4 and GM-CSF. On day 5, pDC were thawed. pDC and monocyte-dervied DC were cultured alone or with UV-irradiated M18 (M18UV) or MV-infected M18 (M18MV) for 18hrs. On day6, DC were cultured with the NYESO-1 specific CD8+ T cell clone during 6hrs in presence of brefeldinA. Cells were then stained with anti-CD8+ antibody, fixed, permeabilized and stained with IFN-g specific antibody. IFN-g secretion by the the T cell clone was analyzed by flow cytometry. We conclude that level of cross-presentation was somewhat similar between the two cell types. It was a little bit weaker for pDC, but these cells have been frozen, whereas Monocyte-derived DC were not. (B) scatter plot representation of three cross-presentation experiments performed with monocyte-derived DC. Error bars represent standard deviation.</p>
Abstract Human papillomavirus (HPV) type 16 infection is one of the most important etiological agents of oropharyngeal squamous cell carcinoma. Patients with HPV-positive squamous cell carcinomas of the head and neck were reported to have a better clinical outcome than patients with HPV-negative tumors. Because HPV16 E6 and E7 oncoproteins are highly immunogenic and constitutively expressed within the tumor cells, HPV-specific T cell immunity may contribute to the better prognosis of HPV-positive tumors. We analyzed the frequency, phenotype and function of HPV16 E6/E7-specific tumor-infiltrating T cells (TILs) in oropharyngeal tumors and tested the effect of anti-PD1 mAb (nivolumab), soluble Tim-3 (sTim-3) and homeostatic in vitro expansion on these characteristics. We show that 73.1% of HPV-associated oropharyngeal tumors are positive for HPV16-specific T cells capable of producing IFNγ upon stimulation with E6/E7 peptide pools. These IFNγ-producing HPV-specific TILs were mainly PD-1+Tim-3-CD8+ T cells, identifying Tim-3 rather than PD-1 as a marker of T cell dysfunction. Consequently, specific IFNγ production was further enhanced by combined nivolumab plus sTim-3 treatment, but not with nivolumab alone. Our data provide the rationale for exploring additional setups of anti-PD-1 mAb treatment, including combinations with other check-point blockers, such as Tim-3 and/or with HPV16-directed therapeutic vaccines. Citation Format: Simona Partlova, Kamila Hladikova, Vladimir Koucky, Anna Fialova, Radek Spisek, Jan Boucek, Michal Zabrodsky, Michael Halaska, Ruth Tachezy, Marek Grega, Jean-François Fonteneau. Dysfunction of HPV16-specific CD8+ T cells derived from oropharyngeal tumors is related to the expression of Tim-3 but not PD-1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1002.
The functional characteristics of CD8+ T cells specific for melanoma antigens (MAs) have often been defined after in vitro culture using nonprofessional antigen-presenting cells. We have examined CD8+ T-cell immunity to MAs and a viral antigen (influenza) in uncultured T cells of healthy donors and melanoma patients using autologous, mature, monocyte-derived dendritic cells (DCs) pulsed with peptide antigens and viral vectors. Antigen-specific IFN-gamma-producing T cells reactive with HLA-A*0201-restricted peptides from four melanoma antigens (MelanA/MART-1, MAGE-3, tyrosinase, and gp100) were detected only at low frequencies (<30 per 2 x 10(5) peripheral blood mononuclear cells for each of the MAs) from HLA-A2.1-positive healthy donors (n = 12) and patients with stages III/IV melanoma (n = 8). Detection of MA-specific, but not influenza matrix peptide (Flu-MP)-specific, T cells required a high concentration (10 microg/ml) of the peptide in this assay. Furthermore, these T cells did not recognize endogenously processed antigen on tumor cell lines or cells infected with viral vectors capable of expressing MAs. The use of autologous, mature DCs led to a significant increase in the number of Flu-MP, but not MA-specific, T cells in 16-h ELISPOT assays for both melanoma patients and healthy donors. In 1-week cocultures with DCs pulsed with 10 microg/ml peptide, MelanA/MART-1-specific T cells did not readily proliferate or differentiate into lytic effectors, in contrast to strong influenza-specific lytic responses. Therefore, despite distinct memory responses to influenza antigens, melanoma patients and healthy controls have a paucity of MA-reactive memory T cells, failing to rapidly generate IFN-gamma-secreting lytic effectors in short-term assays, even when stimulated by DCs.
// Fabien Gueugnon 1,2,3 , Pierre-François Cartron 1,2,3,4 , Cedric Charrier 5 , Philippe Bertrand 4,5 , Jean-François Fonteneau 1,2,3 , Marc Gregoire 1,2,3 and Christophe Blanquart 1,2,3,4 1 Inserm, U892, F-44000, Nantes, France 2 CNRS, UMR6299, F-44000, Nantes, France 3 Université Nantes, F-44000, Nantes, France 4 Réseau Epigénétique du Canceropôle Grand Ouest 5 CNRS, UMR7285, Institut de Chimie des Milieux et Matériaux de Poitiers, Université de Poitiers, France Correspondence: Christophe Blanquart1, email: // Keywords : Mesothelioma, lung cancer, chemotherapy and epigenetic Received : April 2, 2014 Accepted : June 1, 2014 Published : June 3, 2014 Abstract Histone deacetylase inhibitors (HDACi) have shown promising antitumor effects on numerous cancer cells including malignant pleural mesothelioma (MPM) and lung adenocarcinoma (ADCA) cells. However, clinical trials using these compounds alone have shown limited efficacy against solid tumors. Therefore, new molecules are being developed and combinations with classical chemotherapeutic drugs are being tested. Here, we have evaluated on three MPM and three lung ADCA cell lines the antitumor potential of four new HDACi compounds, either alone or in combination with cisplatin. These effects were compared with those of vorinostat, an HDACi approved for cancer treatments. First, we characterized the HDAC mRNA expression profiles of tumor cells and showed an increase of the classI/classII HDAC ratio. We then treated cancer cells with these new HDACi and observed a cell-death induction and an increase of HDACi target genes and proteins expression. This was particularly evident for NODH compound (pan-HDACi) which had similar effects at nanomolar concentrations as micromolar concentrations of vorinostat. Interestingly, we observed that the HDACi/cisplatin combination strongly increased cell-death and limited resistance-phenotype emergence as compared with results obtained when the drugs were used alone. These results could be exploited to develop MPM and lung ADCA treatments combining chemotherapeutic approaches.
Much effort has been made over the last decade to use dendritic cells (DCs) in vaccines to induce specific antitumor immune responses. However, the great hope provided by in vitro and in vivo preclinical investigations was not translated to the clinic in terms of clinical efficacy. Thus, one of the challenges resides in optimizing DC-based therapy to give maximum clinical efficacy while using manufacturing processes that enable quality control and scale-up of consistent products. In this article, we review DC biology and the DC-based clinical trials performed to date and focus on the DC maturation status compatible with the goals of cancer immunotherapy. We also highlight the different approaches used in these clinical studies, such as the DC types or subtypes used and their preparation. Finally, we discuss the immunological and clinical outcomes in treated patients, with emphasis on the strategies that could be used to improve DC-based vaccination.
<p>AVI file - 13330K, Supplemental Figure 2: absence of infection of pDC by MV-eGFP. pDC were cocultured with MV-eGFP (MOI=1) in the presence of IL-3. Fluorescence microscopy video-recording was performed with a picture taken every 15 minutes with a time lapse confocal microscope (Nikon). One cell becomes infected after 12 hours.</p>
The large majority of known melanoma-associated antigenic peptides presented by MHC class I molecules are presented by the most frequent allele, HLA-A*0201. Thus although a significant percentage of Caucasians express HLA-A3, no melanoma-associated antigenic peptide presented by this allele has yet been identified. We show here that the T cell clone M45-10 isolated from tumor infiltrating lymphocytes recovered from a melanoma biopsy recognizes the gp100-derived peptide ALLAVGATK presented by HLA-A*0301. Since gp100 is expressed on most melanoma cells, our results imply that the gp100-based anti-melanoma strategies developed for individuals expressing HLA-A2 will also be applicable to those expressing HLA-AS (about one Caucasian in four). gp100 is therefore a particularly promising melanoma antigen, as different peptides derived from it can be presented by at least two different frequently encountered HLA class I molecules.
Background: Oncolytic viruses such as live-attenuated, vaccine strains of measles virus (MV) have recently emerged as promising cancer treatments, having shown significant antitumor activity against a large variety of human tumors. Objective: Our study aims at determining which parameters define the sensitivity of human melanoma cells to oncolytic MV infection. Methods: We analyzed both in vitro and in vivo the oncolytic activity of MV against a panel of human melanoma cell established in our laboratory. We tested whether either type I interferons or the interferon pathway inhibitor Ruxolitinib could modulate the sensitivity of these cells to oncolytic MV infection. Results: Human melanoma cells exhibit varying levels of sensitivity to MV infection in culture and as tumor xenografts. As these differences are not explained by their expression level of the CD46 receptor, we hypothesized that antiviral immune responses may be suppressed in certain cell resulting in their inability to control infection efficiently. By analyzing the type I IFN response, we found that resistant cells had a fully functional pathway that was activated upon MV infection. On the contrary, sensitive cell showed defects in this pathway. When pre-treated with IFN-α and IFN-β, all but one of the sensitive cell became resistant to MV. Cells resistant to MV were rendered sensitive to MV with Ruxolitinib. Conclusion: Type I interferon response is the main determinant for the sensitivity or resistance of melanoma to oncolytic MV infection. This will have to be taken into account for future clinical trials on oncolytic MV. Keywords: Melanoma, Interferon, Measles virus, Oncolytic, Cancer virotherapy, Ruxolitinib.
