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1.  Signal transducer and activator of transcription 3 in myeloid-derived suppressor cells: an opportunity for cancer therapy 
Oncotarget  2016;7(27):42698-42715.
Cancer progression is in part determined by interactions between cancer cells and stromal cells in the tumor microenvironment (TME). The identification of cytotoxic tumor-infiltrating lymphocytes has instigated research into immune stimulating cancer therapies. Although a promising direction, immunosuppressive mechanisms exerted at the TME hamper its success. Myeloid-derived suppressor cells (MDSCs) have come to the forefront as stromal cells that orchestrate the immunosuppressive TME. Consequently, this heterogeneous cell population has been the object of investigation. Studies revealed that the transcription factor signal transducer and activator of transcription 3 (STAT3) largely dictates the recruitment, activation and function of MDSCs in the TME. Therefore, this review will focus on the role of this key transcription factor during the MDSC's life cycle and on the therapeutic opportunities it offers.
doi:10.18632/oncotarget.8311
PMCID: PMC5173167  PMID: 27029037
MDSC; STAT3; T cell; immunotherapy; radiotherapy
2.  Particle-mediated Intravenous Delivery of Antigen mRNA Results in Strong Antigen-specific T-cell Responses Despite the Induction of Type I Interferon 
Cancer vaccines based on mRNA are extensively studied. The fragile nature of mRNA has instigated research into carriers that can protect it from ribonucleases and as such enable its systemic use. However, carrier-mediated delivery of mRNA has been linked to production of type I interferon (IFN) that was reported to compromise the effectiveness of mRNA vaccines. In this study, we evaluated a cationic lipid for encapsulation of mRNA. The nanometer-sized, negatively charged lipid mRNA particles (LMPs) efficiently transfected dendritic cells and macrophages in vitro. Furthermore, i.v. delivery of LMPs resulted in rapid expression of the mRNA-encoded protein in spleen and liver, predominantly in CD11c+ cells and to a minor extent in CD11b+ cells. Intravenous immunization of mice with LMPs containing ovalbumin, human papilloma virus E7, and tyrosinase-related protein-2 mRNA, either combined or separately, elicited strong antigen-specific T-cell responses. We further showed the production of type I IFNs upon i.v. LMP delivery. Although this decreased the expression of the mRNA-encoded protein, it supported the induction of antigen-specific T-cell responses. These data question the current notion that type I IFNs hamper particle-mediated mRNA vaccines.
doi:10.1038/mtna.2016.38
PMCID: PMC5022130  PMID: 27327138
cytotoxic T lymphocyte; immunotherapy; liposome; mRNA; particle; type I interferon
3.  Intralymphatic mRNA vaccine induces CD8 T-cell responses that inhibit the growth of mucosally located tumours 
Scientific Reports  2016;6:22509.
The lack of appropriate mouse models is likely one of the reasons of a limited translational success rate of therapeutic vaccines against cervical cancer, as rapidly growing ectopic tumours are commonly used for preclinical studies. In this work, we demonstrate that the tumour microenvironment of TC-1 tumours differs significantly depending on the anatomical location of tumour lesions (i.e. subcutaneously, in the lungs and in the genital tract). Our data demonstrate that E7-TriMix mRNA vaccine-induced CD8+ T lymphocytes migrate into the tumour nest and control tumour growth, although they do not express mucosa-associated markers such as CD103 or CD49a. We additionally show that despite the presence of the antigen-specific T cells in the tumour lesions, the therapeutic outcomes in the genital tract model remain limited. Here, we report that such a hostile tumour microenvironment can be reversed by cisplatin treatment, leading to a complete regression of clinically relevant tumours when combined with mRNA immunization. We thereby demonstrate the necessity of utilizing clinically relevant models for preclinical evaluation of anticancer therapies and the importance of a simultaneous combination of anticancer immune response induction with targeting of tumour environment.
doi:10.1038/srep22509
PMCID: PMC4773884  PMID: 26931556
4.  Intratumoral delivery of mRNA: Overcoming obstacles for effective immunotherapy 
Oncoimmunology  2015;4(5):e1005504.
The immunosuppressive tumor microenvironment (TME) is a major obstacle in cancer immunotherapy. Therefore, it has gained attention as a target site. mRNA emerged as a versatile drug class for cancer therapy. We reported that intratumoral administration of mRNA encoding the fusokine Fβ2 supports tumor-specific T-cell immunity. This study provides proof of concept of the use of mRNA to modulate the TME.
doi:10.1080/2162402X.2015.1005504
PMCID: PMC4485825  PMID: 26155403
cancer; CD8+ T cell; dendritic cell; fusokine; immunotherapy; intratumoral; IFNβ; mRNA; TGFβ ; receptor II; CTLs, cytotoxic T lymphocytes; DCs, dendritic cells; Fβ2, a fusokine consisting of IFNβ, fused to the ectodomain of the TGFβ receptor II; IMP, investigational medicinal product; MDSCs, myeloid-derived suppressor cells; TAAs, tumor-associated antigens; TiDCs, tumor-infiltrating DCs; TME, tumor microenvironment
5.  Cancer-Associated Myeloid Regulatory Cells 
Myeloid cells are critically involved in the pathophysiology of cancers. In the tumor microenvironment (TME), they comprise tumor-associated macrophages (TAMs), neutrophils (TANs), dendritic cells, and myeloid-derived suppressor cells, which are further subdivided into a monocytic subset and a granulocytic subset. Some of these myeloid cells, in particular TAMs and TANs, are divided into type 1 or type 2 cells, according to the paradigm of T helper type 1 or type 2 cells. Type 1-activated cells are generally characterized as cells that aid tumor rejection, while all other myeloid cells are shown to favor tumor progression. Moreover, these cells are often at the basis of resistance to various therapies. Much research has been devoted to study the biology of myeloid cells. This endeavor has proven to be challenging, as the markers used to categorize myeloid cells in the TME are not restricted to particular subsets. Also from a functional and metabolic point of view, myeloid cells share many features. Finally, myeloid cells are endowed with a certain level of plasticity, which further complicates studying them outside their environment. In this article, we challenge the exclusive use of cell markers to unambiguously identify myeloid cell subsets in the TME. We further propose to divide myeloid cells into myeloid regulatory or stimulatory cells according to their pro- or antitumor function, because we contend that for therapeutic purposes it is not targeting the cell subsets but rather targeting their protumor traits; hence, myeloid regulatory cells will push antitumor immunotherapy to the next level.
doi:10.3389/fimmu.2016.00113
PMCID: PMC4810015  PMID: 27065074
cancer; tumor microenvironment; myeloid cells; monocyte; macrophage; dendritic cell; neutrophil; myeloid-derived suppressor cell
6.  Consensus guidelines for the detection of immunogenic cell death 
Kepp, Oliver | Senovilla, Laura | Vitale, Ilio | Vacchelli, Erika | Adjemian, Sandy | Agostinis, Patrizia | Apetoh, Lionel | Aranda, Fernando | Barnaba, Vincenzo | Bloy, Norma | Bracci, Laura | Breckpot, Karine | Brough, David | Buqué, Aitziber | Castro, Maria G. | Cirone, Mara | Colombo, Maria I. | Cremer, Isabelle | Demaria, Sandra | Dini, Luciana | Eliopoulos, Aristides G. | Faggioni, Alberto | Formenti, Silvia C. | Fučíková, Jitka | Gabriele, Lucia | Gaipl, Udo S. | Galon, Jérôme | Garg, Abhishek | Ghiringhelli, François | Giese, Nathalia A. | Guo, Zong Sheng | Hemminki, Akseli | Herrmann, Martin | Hodge, James W. | Holdenrieder, Stefan | Honeychurch, Jamie | Hu, Hong-Min | Huang, Xing | Illidge, Tim M. | Kono, Koji | Korbelik, Mladen | Krysko, Dmitri V. | Loi, Sherene | Lowenstein, Pedro R. | Lugli, Enrico | Ma, Yuting | Madeo, Frank | Manfredi, Angelo A. | Martins, Isabelle | Mavilio, Domenico | Menger, Laurie | Merendino, Nicolò | Michaud, Michael | Mignot, Gregoire | Mossman, Karen L. | Multhoff, Gabriele | Oehler, Rudolf | Palombo, Fabio | Panaretakis, Theocharis | Pol, Jonathan | Proietti, Enrico | Ricci, Jean-Ehrland | Riganti, Chiara | Rovere-Querini, Patrizia | Rubartelli, Anna | Sistigu, Antonella | Smyth, Mark J. | Sonnemann, Juergen | Spisek, Radek | Stagg, John | Sukkurwala, Abdul Qader | Tartour, Eric | Thorburn, Andrew | Thorne, Stephen H. | Vandenabeele, Peter | Velotti, Francesca | Workenhe, Samuel T. | Yang, Haining | Zong, Wei-Xing | Zitvogel, Laurence | Kroemer, Guido | Galluzzi, Lorenzo
Oncoimmunology  2014;3(9):e955691.
Apoptotic cells have long been considered as intrinsically tolerogenic or unable to elicit immune responses specific for dead cell-associated antigens. However, multiple stimuli can trigger a functionally peculiar type of apoptotic demise that does not go unnoticed by the adaptive arm of the immune system, which we named “immunogenic cell death” (ICD). ICD is preceded or accompanied by the emission of a series of immunostimulatory damage-associated molecular patterns (DAMPs) in a precise spatiotemporal configuration. Several anticancer agents that have been successfully employed in the clinic for decades, including various chemotherapeutics and radiotherapy, can elicit ICD. Moreover, defects in the components that underlie the capacity of the immune system to perceive cell death as immunogenic negatively influence disease outcome among cancer patients treated with ICD inducers. Thus, ICD has profound clinical and therapeutic implications. Unfortunately, the gold-standard approach to detect ICD relies on vaccination experiments involving immunocompetent murine models and syngeneic cancer cells, an approach that is incompatible with large screening campaigns. Here, we outline strategies conceived to detect surrogate markers of ICD in vitro and to screen large chemical libraries for putative ICD inducers, based on a high-content, high-throughput platform that we recently developed. Such a platform allows for the detection of multiple DAMPs, like cell surface-exposed calreticulin, extracellular ATP and high mobility group box 1 (HMGB1), and/or the processes that underlie their emission, such as endoplasmic reticulum stress, autophagy and necrotic plasma membrane permeabilization. We surmise that this technology will facilitate the development of next-generation anticancer regimens, which kill malignant cells and simultaneously convert them into a cancer-specific therapeutic vaccine.
doi:10.4161/21624011.2014.955691
PMCID: PMC4292729  PMID: 25941621
ATP release; autophagy; calreticulin; endoplasmic reticulum stress; HMGB1; immunotherapy; APC, antigen-presenting cell; ATF6, activating transcription factor 6; BAK1, BCL2-antagonist/killer 1; BAX, BCL2-associated X protein; BCL2, B-cell CLL/lymphoma 2 protein; CALR, calreticulin; CTL, cytotoxic T lymphocyte; Δψm, mitochondrial transmembrane potential; DAMP, damage-associated molecular pattern; DAPI, 4′,6-diamidino-2-phenylindole; DiOC6(3), 3,3′-dihexyloxacarbocyanine iodide; EIF2A, eukaryotic translation initiation factor 2A; ER, endoplasmic reticulum; FLT3LG, fms-related tyrosine kinase 3 ligand; G3BP1, GTPase activating protein (SH3 domain) binding protein 1; GFP, green fluorescent protein; H2B, histone 2B; HMGB1, high mobility group box 1; HSP, heat shock protein; HSV-1, herpes simplex virus type I; ICD, immunogenic cell death; IFN, interferon; IL, interleukin; MOMP, mitochondrial outer membrane permeabilization; PDIA3, protein disulfide isomerase family A; member 3; PI, propidium iodide; RFP, red fluorescent protein; TLR, Toll-like receptor; XBP1, X-box binding protein 1
7.  Pros and Cons of Antigen-Presenting Cell Targeted Tumor Vaccines 
Journal of Immunology Research  2015;2015:785634.
In therapeutic antitumor vaccination, dendritic cells play the leading role since they decide if, how, when, and where a potent antitumor immune response will take place. Since the disentanglement of the complexity and merit of different antigen-presenting cell subtypes, antitumor immunotherapeutic research started to investigate the potential benefit of targeting these subtypes in situ. This review will discuss which antigen-presenting cell subtypes are at play and how they have been targeted and finally question the true meaning of targeting antitumor-based vaccines.
doi:10.1155/2015/785634
PMCID: PMC4637118  PMID: 26583156
8.  Anti-melanoma vaccines engineered to simultaneously modulate cytokine priming and silence PD-L1 characterized using ex vivo myeloid-derived suppressor cells as a readout of therapeutic efficacy 
Oncoimmunology  2014;3(7):e945378.
Efficacious antitumor vaccines strongly stimulate cancer-specific effector T cells and counteract the activity of tumor-infiltrating immunosuppressive cells. We hypothesised that combining cytokine expression with silencing programmed cell death ligand 1 (PD-L1) could potentiate anticancer immune responses of lentivector vaccines. Thus, we engineered a collection of lentivectors that simultaneously co-expressed an antigen, a PD-L1-silencing shRNA, and various T cell-polarising cytokines, including interferon γ (IFNγ), transforming growth factor β (TGFβ) or interleukins (IL12, IL15, IL23, IL17A, IL6, IL10, IL4). In a syngeneic B16F0 melanoma model and using tyrosinase related protein 1 (TRP1) as a vaccine antigen, we found that simultaneous delivery of IL12 and a PD-L1-silencing shRNA was the only combination that exhibited therapeutically relevant anti-melanoma activities. Mechanistically, we found that delivery of the PD-L1 silencing construct boosted T cell numbers, inhibited in vivo tumor growth and strongly cooperated with IL12 cytokine priming and antitumor activities. Finally, we tested the capacities of our vaccines to counteract tumor-infiltrating myeloid-derived suppressor cell (MDSC) activities ex vivo. Interestingly, the lentivector co-expressing IL12 and the PD-L1 silencing shRNA was the only one that counteracted MDSC suppressive activities, potentially underlying the observed anti-melanoma therapeutic benefit. We conclude that (1) evaluation of vaccines in healthy mice has no significant predictive value for the selection of anticancer treatments; (2) B16 cells expressing xenoantigens as a tumor model are of limited value; and (3) vaccines which inhibit the suppressive effect of MDSC on T cells in our ex vivo assay show promising and relevant antitumor activities.
doi:10.4161/21624011.2014.945378
PMCID: PMC4355828  PMID: 25954597
immunotherapy; MDSC; melanoma; PD-L1; T cell; DC, dendritic cell; G-MDSC, granulocytic MDSC; IL, interleukin; IiOVA, MHC II invariant chain-ovalbumin; MDSC, myeloid-derived suppressor cell; M-MDS, monocytic MDSC; MLR, mixed lymphocyte reaction; OVA, chicken ovalbumin; p1, PD-L1-targeted microRNA; PD-L1, programmed cell death 1 ligand 1; PD-1, programmed cell death 1; shRNA, short hairpin RNA; TAA, tumor associated antigen; TCR, T cell receptor; Th, T helper lymphocyte; TRP1, tyrosinase related protein 1;; TRP2, tyrosinase related protein 2; 142 3p, target sequence for the microRNA 142 3p
9.  Ex vivo generation of myeloid-derived suppressor cells that model the tumor immunosuppressive environment in colorectal cancer 
Oncotarget  2015;6(14):12369-12382.
Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of cells that accumulate in tumor-bearing subjects and which strongly inhibit anti-cancer immune responses. To study the biology of MDSC in colorectal cancer (CRC), we cultured bone marrow cells in conditioned medium from CT26 cells, which are genetically modified to secrete high levels of granulocyte-macrophage colony-stimulating factor. This resulted in the generation of high numbers of CD11b+ Ly6G+ granulocytic and CD11b+ Ly6C+ monocytic MDSC, which closely resemble those found within the tumor but not the spleen of CT26 tumor-bearing mice. Such MDSC potently inhibited T-cell responses in vitro, a process that could be reversed upon blocking of arginase-1 or inducible nitric oxide synthase (iNOS). We confirmed that inhibition of arginase-1 or iNOS in vivo resulted in the stimulation of cytotoxic T-cell responses. A delay in tumor growth was observed upon functional repression of both enzymes. These data confirm the role of MDSC as inhibitors of T-cell-mediated immune responses in CRC. Moreover, MDSC differentiated in vitro from bone marrow cells using conditioned medium of GM-CSF-secreting CT26 cells, represent a valuable platform to study/identify drugs that counteract MDSC activities.
PMCID: PMC4494944  PMID: 25869209
MDSC; CRC; arginase-1; inducible nitric oxide synthase; GM-CSF
10.  Targeting the tumor microenvironment to enhance antitumor immune responses 
Oncotarget  2014;6(3):1359-1381.
The identification of tumor-specific antigens and the immune responses directed against them has instigated the development of therapies to enhance antitumor immune responses. Most of these cancer immunotherapies are administered systemically rather than directly to tumors. Nonetheless, numerous studies have demonstrated that intratumoral therapy is an attractive approach, both for immunization and immunomodulation purposes. Injection, recruitment and/or activation of antigen-presenting cells in the tumor nest have been extensively studied as strategies to cross-prime immune responses. Moreover, delivery of stimulatory cytokines, blockade of inhibitory cytokines and immune checkpoint blockade have been explored to restore immunological fitness at the tumor site. These tumor-targeted therapies have the potential to induce systemic immunity without the toxicity that is often associated with systemic treatments. We review the most promising intratumoral immunotherapies, how these affect systemic antitumor immunity such that disseminated tumor cells are eliminated, and which approaches have been proven successful in animal models and patients.
PMCID: PMC4359300  PMID: 25682197
Intratumoral; Immunotherapy; Tumor microenvironment; Immunomodulation; Vaccination
11.  Combinatorial Strategies for the Induction of Immunogenic Cell Death 
The term “immunogenic cell death” (ICD) is commonly employed to indicate a peculiar instance of regulated cell death (RCD) that engages the adaptive arm of the immune system. The inoculation of cancer cells undergoing ICD into immunocompetent animals elicits a specific immune response associated with the establishment of immunological memory. Only a few agents are intrinsically endowed with the ability to trigger ICD. These include a few chemotherapeutics that are routinely employed in the clinic, like doxorubicin, mitoxantrone, oxaliplatin, and cyclophosphamide, as well as some agents that have not yet been approved for use in humans. Accumulating clinical data indicate that the activation of adaptive immune responses against dying cancer cells is associated with improved disease outcome in patients affected by various neoplasms. Thus, novel therapeutic regimens that trigger ICD are urgently awaited. Here, we discuss current combinatorial approaches to convert otherwise non-immunogenic instances of RCD into bona fide ICD.
doi:10.3389/fimmu.2015.00187
PMCID: PMC4408862  PMID: 25964783
ATP; autophagy; calreticulin; endoplasmic reticulum stress; HMGB1; type I interferon
12.  Corrigendum: “Combinatorial Strategies for the Induction of Immunogenic Cell Death” 
doi:10.3389/fimmu.2015.00275
PMCID: PMC4450300  PMID: 26082782
ATP; autophagy; calreticulin; endoplasmic reticulum stress; HMGB1 protein; type I interferon
13.  Molecular and Translational Classifications of DAMPs in Immunogenic Cell Death 
The immunogenicity of malignant cells has recently been acknowledged as a critical determinant of efficacy in cancer therapy. Thus, besides developing direct immunostimulatory regimens, including dendritic cell-based vaccines, checkpoint-blocking therapies, and adoptive T-cell transfer, researchers have started to focus on the overall immunobiology of neoplastic cells. It is now clear that cancer cells can succumb to some anticancer therapies by undergoing a peculiar form of cell death that is characterized by an increased immunogenic potential, owing to the emission of the so-called “damage-associated molecular patterns” (DAMPs). The emission of DAMPs and other immunostimulatory factors by cells succumbing to immunogenic cell death (ICD) favors the establishment of a productive interface with the immune system. This results in the elicitation of tumor-targeting immune responses associated with the elimination of residual, treatment-resistant cancer cells, as well as with the establishment of immunological memory. Although ICD has been characterized with increased precision since its discovery, several questions remain to be addressed. Here, we summarize and tabulate the main molecular, immunological, preclinical, and clinical aspects of ICD, in an attempt to capture the essence of this phenomenon, and identify future challenges for this rapidly expanding field of investigation.
doi:10.3389/fimmu.2015.00588
PMCID: PMC4653610  PMID: 26635802
anti-tumor immunity; immunogenicity; immunotherapy; molecular medicine; oncoimmunology; patient prognosis; translational medicine
14.  Intratumoral administration of mRNA encoding a fusokine consisting of IFN-β and the ectodomain of the TGF-β receptor II potentiates antitumor immunity 
Oncotarget  2014;5(20):10100-10113.
It is generally accepted that the success of immunotherapy depends on the presence of tumor-specific CD8+ cytotoxic T cells and the modulation of the tumor environment. In this study, we validated mRNA encoding soluble factors as a tool to modulate the tumor microenvironment to potentiate infiltration of tumor-specific T cells. Intratumoral delivery of mRNA encoding a fusion protein consisting of interferon-β and the ectodomain of the transforming growth factor-β receptor II, referred to as Fβ2, showed therapeutic potential. The treatment efficacy was dependent on CD8+ T cells and could be improved through blockade of PD-1/PD-L1 interactions. In vitro studies revealed that administration of Fβ2 to tumor cells resulted in a reduced proliferation and increased expression of MHC I but also PD-L1. Importantly, Fβ2 enhanced the antigen presenting capacity of dendritic cells, whilst reducing the suppressive activity of myeloid-derived suppressor cells. In conclusion, these data suggest that intratumoral delivery of mRNA encoding soluble proteins, such as Fβ2, can modulate the tumor microenvironment, leading to effective antitumor T cell responses, which can be further potentiated through combination therapy.
PMCID: PMC4259408  PMID: 25338019
mRNA; IFN-β; TGF-β; cancer therapy; T cell
15.  A highly efficient tumor-infiltrating MDSC differentiation system for discovery of anti-neoplastic targets, which circumvents the need for tumor establishment in mice 
Oncotarget  2014;5(17):7843-7857.
Myeloid-derived suppressor cells (MDSCs) exhibit potent immunosuppressive activities in cancer. MDSCs infiltrate tumors and strongly inhibit cancer-specific cytotoxic T cells. Their mechanism of differentiation and identification of MDSC-specific therapeutic targets are major areas of interest. We have devised a highly efficient and rapid method to produce very large numbers of melanoma-infiltrating MDSCs ex vivo without inducing tumors in mice. These MDSCs were used to study their differentiation, immunosuppressive activities and were compared to non-neoplastic counterparts and conventional dendritic cells using unbiased systems biology approaches. Differentially activated/deactivated pathways caused by cell type differences and by the melanoma tumor environment were identified. MDSCs increased the expression of trafficking receptors to sites of inflammation, endocytosis, changed lipid metabolism, and up-regulated detoxification pathways such as the expression of P450 reductase. These studies uncovered more than 60 potential novel therapeutic targets. As a proof of principle, we demonstrate that P450 reductase is the target of pro-drugs such as Paclitaxel, which depletes MDSCs following chemotherapy in animal models of melanoma and in human patients. Conversely, P450 reductase protects MDSCs against the cytotoxic actions of other chemotherapy drugs such as Irinotecan, which is ineffective for the treatment of melanoma.
PMCID: PMC4202165  PMID: 25151659
16.  Targeting of Human Antigen-Presenting Cell Subsets 
Journal of Virology  2013;87(20):11304-11308.
Antigen-presenting cells are a heterogeneous group of cells that are characterized by their functional specialization. Consequently, targeting specific antigen-presenting cell subsets offers opportunities to induce distinct T cell responses. Here we report on the generation and use of nanobodies (Nbs) to target lentivectors specifically to human lymph node-resident myeloid dendritic cells, demonstrating that Nbs represent a powerful tool to redirect lentivectors to human antigen-presenting cell subsets.
doi:10.1128/JVI.01498-13
PMCID: PMC3807283  PMID: 23864630
17.  Immunogenicity of targeted lentivectors 
Oncotarget  2014;5(3):704-715.
To increase the safety and possibly efficacy of HIV-1 derived lentivectors (LVs) as an anti-cancer vaccine, we recently developed the Nanobody (Nb) display technology to target LVs to antigen presenting cells (APCs). In this study, we extend these data with exclusive targeting of LVs to conventional dendritic cells (DCs), which are believed to be the main cross-presenting APCs for the induction of a TH1-conducted antitumor immune response. The immunogenicity of these DC-subtype targeted LVs was compared to that of broad tropism, general APC-targeted and non-infectious LVs. Intranodal immunization with ovalbumin encoding LVs induced proliferation of antigen specific CD4+ T cells, irrespective of the LVs' targeting ability. However, the cytokine secretion profile of the restimulated CD4+ T cells demonstrated that general APC targeting induced a similar TH1-profile as the broad tropism LVs while transduction of conventional DCs alone induced a similar and less potent TH1 profile as the non-infectious LVs. This observation contradicts the hypothesis that conventional DCs are the most important APCs and suggests that the activation of other APCs is also meaningful. Despite these differences, all targeted LVs were able to stimulate cytotoxic T lymphocytes, be it to a lesser extent than broad tropism LVs. Furthermore this induction was shown to be dependent on type I interferon for the targeted and non-infectious LVs, but not for broad tropism LVs. Finally we demonstrated that the APC-targeted LVs were as potent in therapy as broad tropism LVs and as such deliver on their promise as safer and efficacious LV-based vaccines.
PMCID: PMC3996667  PMID: 24519916
lentivector; targeting; antigen presenting cell; vaccine; antitumor immunotherapy
18.  Design of an Optimized Wilms' Tumor 1 (WT1) mRNA Construct for Enhanced WT1 Expression and Improved Immunogenicity In Vitro and In Vivo 
Tumor antigen–encoding mRNA for dendritic cell (DC)-based vaccination has gained increasing popularity in recent years. Within this context, two main strategies have entered the clinical trial stage: the use of mRNA for ex vivo antigen loading of DCs and the direct application of mRNA as a source of antigen for DCs in vivo. DCs transfected with mRNA-encoding Wilms' tumor 1 (WT1) protein have shown promising clinical results. Using a stepwise approach, we re-engineered a WT1 cDNA-carrying transcription vector to improve the translational characteristics and immunogenicity of the transcribed mRNA. Different modifications were performed: (i) the WT1 sequence was flanked by the lysosomal targeting sequence of dendritic cell lysosomal-associated membrane protein to enhance cytoplasmic expression; (ii) the nuclear localization sequence (NLS) of WT1 was deleted to promote shuttling from the nucleus to the cytoplasm; (iii) the WT1 DNA sequence was optimized in silico to improve translational efficiency; and (iv) this WT1 sequence was cloned into an optimized RNA transcription vector. DCs electroporated with this optimized mRNA showed an improved ability to stimulate WT1-specific T-cell immunity. Furthermore, in a murine model, we were able to show the safety, immunogenicity, and therapeutic activity of this optimized mRNA. This work is relevant for the future development of improved mRNA-based vaccine strategies K.
doi:10.1038/mtna.2013.54
PMCID: PMC3889186  PMID: 24253259
19.  Role of non-classical MHC class I molecules in cancer immunosuppression 
Oncoimmunology  2013;2(11):e26491.
Growing neoplasms employ various mechanisms to evade immunosurveillance. The expression of non-classical MHC class I molecules by both immune and malignant cells in the tumor microenvironment constitute of the strategies used by tumors to circumvent the cytotoxic activity of effector cells of the immune system. The overexpression of HLA-G, -E, and -F is a common finding across a variety of malignancies. However, while the presence of HLA-G and HLA-E has been recently correlated with poor clinical outcome, information on the clinicopathological significance of HLA-F is limited. In the present review, we summarize studies on non-classical MHC class I molecules with special emphasis on their role in the modulation of anticancer immune responses.
doi:10.4161/onci.26491
PMCID: PMC3894240  PMID: 24482746
antigen presentation; cancer; immunomodulation; NK cells; non-classical MHC class I molecules; T cells
20.  Assessing T-cell responses in anticancer immunotherapy 
Oncoimmunology  2013;2(10):e26148.
Since dendritic cells operate as professional antigen-presenting cells (APCs) and hence are capable of jumpstarting the immune system, they have been exploited to develop a variety of immunotherapeutic regimens against cancer. In the few past years, myeloid-derived suppressor cells (MDSCs) have been shown to mediate robust immunosuppressive functions, thereby inhibiting tumor-targeting immune responses. Thus, we propose that the immunomodulatory activity of MDSCs should be carefully considered for the development of efficient anticancer immunotherapies.
doi:10.4161/onci.26148
PMCID: PMC3825722  PMID: 24244902
antigen presentation; cancer; dendritic cells; myeloid-derived suppressor cell; T cells
21.  Preclinical Evaluation of Invariant Natural Killer T Cells in the 5T33 Multiple Myeloma Model 
PLoS ONE  2013;8(5):e65075.
Immunomodulators have been used in recent years to reactivate host anti-tumor immunity in several hematological malignancies. This report describes the effect of activating natural killer T (NKT) cells by α-Galactosylceramide (α-GalCer) in the 5T33MM model of multiple myeloma (MM). NKT cells are T lymphocytes, co-expressing T and NK receptors, while invariant NKT cells (iNKTs) also express a unique semi-invariant TCR α-chain. We followed iNKT numbers during the development of the disease in both 5T33MM mice and MM patients and found that their numbers dropped dramatically at the end stage of the disease, leading to a loss of total IFN-γ secretion. We furthermore observed that α-GalCer treatment significantly increased the survival of 5T33MM diseased mice. Taken together, our data demonstrate for the first time the possibility of using a preclinical murine MM model to study the effects of α-GalCer and show promising results of α-GalCer treatment in a low tumor burden setting.
doi:10.1371/journal.pone.0065075
PMCID: PMC3669090  PMID: 23741460
22.  Retroviral and lentiviral vectors for the induction of immunological tolerance 
Scientifica  2012;2012:694137.
doi:10.6064/2012/694137
PMCID: PMC3605697  PMID: 23526794
Autoimmune disease; arthritis; co-stimulation; dendritic cell; immunotherapy; signalling; T cell; tolerance; MAPK; NF-κB; microRNA
23.  PD-L1/PD-1 Co-Stimulation, a Brake for T cell Activation and a T cell Differentiation Signal 
For T cell activation, three signals have to be provided from the antigen presenting cell; Signal 1 (antigen recognition), signal 2 (co-stimulation) and signal 3 (cytokine priming). Blocking negative co-stimulation during antigen presentation to T cells is becoming a promising therapeutic strategy to enhance cancer immunotherapy. Here we will focus on interference with PD-1/PD-L1 negative co-stimulation during antigen presentation to T cells as a therapeutic approach. We will discuss the potential mechanisms and the therapeutic consequences by which interference/inhibition with this interaction results in anti-tumour immunity. Particularly, we will comment on whether blocking negative co-stimulation provides differentiation signals to T cells undergoing antigen presentation. A major dogma in immunology states that T cell differentiation signals are given by cytokines and chemokines (signal 3) rather than co-stimulation (signal 2). We will discuss whether this is the case when blocking PD-L1/PD-1 negative co-stimulation.
doi:10.4172/2155-9899.S12-006
PMCID: PMC3605779  PMID: 23525238
Cancer; Co-stimulation; Dendritic cell; Immunotherapy; PD-L1; PD-1; CD80
24.  MODULATING CO-STIMULATION DURING ANTIGEN PRESENTATION TO ENHANCE CANCER IMMUNOTHERAPY 
One of the key roles of the immune system is the identification of potentially dangerous pathogens or tumour cells, and raising a wide range of mechanisms to eliminate them from the organism. One of these mechanisms is activation and expansion of antigen-specific cytotoxic T cells, after recognition of antigenic peptides on the surface of antigen presenting cells such as dendritic cells (DCs). However, DCs also process and present autoantigens. Therefore, antigen presentation has to occur in the appropriate context to either trigger immune responses or establishing immunological tolerance. This is achieved by co-stimulation of T cells during antigen presentation. Co-stimulation consists on the simultaneous binding of ligand-receptor molecules at the immunological synapse which will determine the type and extent of T cell responses. In addition, the type of cytokines/chemokines present during antigen presentation will influence the polarisation of T cell responses, whether they lead to tolerance, antibody responses or cytotoxicity. In this review, we will focus on approaches manipulating co-stimulation during antigen presentation, and the role of cytokine stimulation on effective T cell responses. More specifically, we will address the experimental strategies to interfere with negative co-stimulation such as that mediated by PD-L1 (Programmed cell death 1 ligand 1)/PD-1 (Programmed death 1) to enhance anti-tumour immunity.
PMCID: PMC3428911  PMID: 22945252
Cancer; Co-stimulation; Dendritic cell; Immunotherapy; Signalling; T cell; B7; PD-L1; PD-L2; CTLA4; PD-1; CD80; MAPK; NF-κB
25.  mRNA 
Two decades ago, mRNA became the focus of research in molecular medicine and was proposed as an active pharmaceutical ingredient for the therapy of cancer. In this regard, mRNA has been mainly used for ex vivo modification of antigen-presenting cells (APCs), such as dendritic cells (DCs). This vaccination strategy has proven to be safe, well tolerated and capable of inducing tumor antigen-specific immune responses. Recently, the direct application of mRNA for in situ modification of APCs, hence immunization was shown to be feasible and at least as effective as DC-based immunization in pre-clinical models. It is believed that application of mRNA as an off-the-shelf vaccine represents an important step in the development of future cancer immunotherapeutic strategies. Here, we will discuss the use of ex vivo mRNA-modified DCs and “naked mRNA” for cancer immunotherapy focusing on parameters such as the employed DC subtype, DC activation stimulus and route of immunization. In addition, we will provide an overview on the clinical trials published so far, trying to link their outcome to the aforementioned parameters.
doi:10.4161/hv.22661
PMCID: PMC3859745  PMID: 23291946
mRNA; dendritic cell; immunotherapy; cancer

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