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The II melanoma translational meeting of the Spanish Melanoma Group (GEM) was held in Barcelona, Spain, at the Hospital Universitari Quiron-Dexeus on November 3 rd , 2016. The conference featured leaders in the fields of oncology, immunology and dermatology, all working at both national and international levels on basic research with direct applications to the clinical setting. The objective was to present cutting edge research on melanoma, mainly from Spanish research groups, but also from some international groups, with the aim of generating a network for future national and international collaborations. During a single day, fifteen speakers, seven biologists and eight clinicians, with a special focus on translational research, outlined the main findings of their ongoing studies. The moderators in every session were recognized GEM oncologists who guided discussion from the clinical point of view regarding the preclinical data presented. Herein, we summarize the main topics discussed during the meeting.

Desde comienzos del siglo XX, los científicos han intentado aprovechar las actividades naturales del sistema inmunológico para curar el cáncer. Sin embargo, las inmunoterapias no han dado el resultado clínico que podría haberse esperado. De hecho, lo tratamientos anti-neoplásicos clásicos como la cirugía, la radioterapia y la quimioterapia siguen consistiendo en la primera línea de tratamiento. Aun así, existe un gran número de evidencias experimentales sobre la inmunogenicidad de las células cancerosas. Sin embargo, la activación efectiva de las respuestas T anti-cancerosas depende estrechamente de la presentación eficiente de antígenos tumorales por parte de células presentadoras de antígeno profesionales, como las células dendríticas (dendritic cells, DC). Aunque se han desarrollado un gran número de estrategias para reforzar las funciones de presentación de antígeno de las DC, la inmunoterapia como tratamiento anti-neoplásico todavía no es tan efectiva como esperaríamos de acuerdo con los resultados obtenidos en modelos preclínicos durante las últimas décadas. En este trabajo no pretendemos revisar exhaustivamente la inmunoterapia con DC, un campo ampliamente extenso y tratado en otras revisiones especializadas. Aquí se exponen la experiencias que nuestro grupo ha llevado a cabo durante la última década modificando genéticamente a las DC para mejorar su eficacia anti-tumoral. Since the beginning of the 20th century, biomedical scientists have tried to take advantage of the natural anti-cancer activities of the immune system. However, all the scientific and medical efforts dedicated to this have not resulted in the expected success. In fact, classical antineoplastic treatments such as surgery, radio and chemotherapy are still first line treatments. Even so, there is a quantity of experimental evidence demonstrating that cancer cells are immunogenic. However, the effective activation of anti-cancer T cell responses closely depends on an efficient antigen presentation carried out by professional antigen presenting cells such as DC. Although there are a number of strategies to strengthen antigen presentation by DC, anti-cancer immunotherapy is not as effective as we would expect according to preclinical data accumulated in recent decades. We do not aim to make an exhaustive review of DC immunotherapy here, which is an extensive research subject already dealt with in many specialised reviews. Instead, we present the experimental approaches undertaken by our group over the last decade, by modifying DC to improve their anti-tumour capacities.

Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of cells that accumulate in tumor bearing subjects and amongst others inhibit immune responses. To study the biology of MDSC in the context of colorectal cancer (CRC), we cultured bone marrow cells in conditioned medium of the cell line CT26 that was modified to secrete high levels of granulocyte-macrophage colony-stimulating factor. This resulted in the generation of CD11b+ Ly6G+ granulocytic and CD11b+ Ly6C+ monocytic MDSC, which closely resemble those found within the tumor but not the spleen of CT26 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 next showed that inhibition of arginase-1 or iNOS in vivo resulted in the stimulation of cytotoxic T-cell responses. A delay in tumor growth could be observed as long as both enzymes were functionally repressed. These data confirm the role of MDSC as inhibitors of T cell-mediated immune responses in CRC. Moreover, our data show that bone marrow cells differentiated in vitro using conditioned medium of GM-CSF secreting CT26 cells, represent a valuable platform to study drugs that are developed to counteract the activity of MDSC.

Immunotherapy for treatment of cancer is emerging as an alternative that overcomes the limitations of classical therapies. However a recurring issue in immunotherapy is the immunosuppressive tumor environment, which hampers antitumor T cell responses. Since we previously demonstrated the feasibility of delivering mRNA to the tumor, we propose to deliver mRNA encoding F?2, a fusion protein consisting of IFN-? and the extracellular part of the TGF-?II receptor to revert this hostile environment. To demonstrate that F?2 mRNA was translated into a functional protein we electroporated 293T cells. Supernatants collected 24 hours after electroporation were evaluated in several reporter assays. Next, we evaluated the effect of the fusion protein on tumor cells, T cells and myeloid cells. We demonstrated in vitro that treatment of tumor cells with F?2 results in up-regulation of MHC class I and reduced tumor cell proliferation. In vitro treatment of dendritic cells with the fusion protein resulted in their maturation and enhanced ability to activate ovalbumin-specific CD8+ T cells. In addition, in vitro treatment of T cells with F?2 resulted in enhanced production of IFN-?. We also investigated the effect of the F?2 fusokine on myeloid-derived suppressor cells. Exposure of these cells to F?2 resulted in changes in their phenotype and suppressive capacity as T cells retained their ability to produce IFN-?. Encouraged by these results, we treated mice bearing palpable ovalbumin-expressing tumors (E.G7-OVA) with single or repeated injections of F?2 or control mRNA. We demonstrated that a single injection of F?2 mRNA resulted in a short-term delay of tumor growth, which could be prolonged by repeated injections of high doses of the mRNA. Taken together, our results demonstrate the feasibility of using mRNA encoding the immune modulating protein F?2 to engineer the tumor environment. These results highlight the prospective for the use of mRNA to achieve therapeutic antitumor immunity.

T cell receptor (TCR) down-modulation after antigen presentation is a fundamental process that regulates TCR signal transduction. Current understanding of this process is that intrinsic TCR/CD28 signal transduction leads to TCR down-modulation. We demonstrate a novel mechanism whereby the interaction between programmed cell death 1 ligand 1 (PD-L1) on dendritic cells (DCs) and programmed death 1 (PD-1) on CD8 T cells triggers ligand-induced TCR down-modulation. This occurred via Cbl-b E3 up-regulation in CD8 T cells. PD-L1 silencing in DCs with lentiviral vectors delivering microRNAs markedly inhibited TCR down-modulation. PD-L1 silencing hyper-activated CD8 T cells as assessed in vitro and in vivo in an arthritis model. Antitumour immune responses (lymphoma and melanoma) were accelerated and DC anti-tumour capacities potentiated, when combined with mitogen-activated kinase (MAPK) modulators that promote DC activation, or with cytokine priming.

Dendritic cells (DCs) are the most potent professional antigen presenting cells (APCs) of the immune system. DCs provide three different signals to activate effector T cells during antigen presentation. Signal 1 is delivered by the binding of the T cell receptor (TCR) to the peptide antigen complexed to MHC molecules, which is exposed on the surface of DCs. Signal 2 is provided by the integration of positive and negative co-stimulatory interactions on the surfaces of DCs and T cells. In order to direct the differentiation of T cells into different subtypes a third signal is induced by the secretion of cytokines by the DCs. To enhance cancer immunotherapy we have developed a lentivector, which simultaneously modulates these three signals. First, an antigen of interest is included in the lentivector to provide signal 1. Second, a PD-L1-targeted shRNA that eliminates PD-L1 expression on DCs reinforces positive co-stimulation (signal 2). And finally a cytokine array to modulate signal 3 is expressed to induce thedesired anti-cancer effector T cell response. Using this lentivector expression system, we expressed a collection of Th1-, Th2-, Th17-, and Treg-inducing cytokines and evaluated their antigenicity in cultured DCs. We also evaluated these lentivectors by vaccination in mice and found that we could induce distinct T cell profiles combined with low Treg numbers. Finally, we have data demonstrating anti-tumour activities of some of these constructs in a mouse model for melanoma. Concluding, we have developed a lentivector system that is capable of activating as well as fine-tuning adoptive T cell responses according to the disease in question and that induces in vivo anti-tumour responses in mice.

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.

Delivery of tumour-associated antigens (TAA) in a way that induces effective, specific immunity is a challenge in anti-cancer vaccine design. Circumventing tumour-induced tolerogenic mechanisms in vivo is also critical for effective immunotherapy. Effective immune responses are induced by professional antigen presenting cells, in particular dendritic cells (DC). This requires presentation of the antigen to both CD4(+) and CD8(+) T cells in the context of strong co-stimulatory signals. Lentiviral vectors have been tested as vehicles, for both ex vivo and in vivo delivery of TAA and/or activation signals to DC, and have been demonstrated to induce potent T cell mediated immune responses that can control tumour growth. This review will focus on the use of lentiviral vectors for in vivo gene delivery to DC, introducing strategies to target DC, either targeting cell entry or gene expression to improve safety of the lentiviral vaccine or targeting dendritic cell activation pathways to enhance performance of the lentiviral vaccine. In conclusion, this review highlights the potential of lentiviral vectors as a generally applicable 'off-the-shelf' anti-cancer immunotherapeutic.

Lentiviral vectors are promising vaccine vector candidates, which have been extensively tested in pre-clinical models of infectious disease and cancer immunotherapy. They are also used in gene therapy clinical trials, both for the ex vivo modification of cells and for direct in vivo injection. It is therefore critical to understand the mechanism(s) by which such vectors might stimulate the immune system. We evaluated the effect of lentiviral vectors on myeloid dendritic cells (DC), the main target of lentiviral transduction following subcutaneous immunisation. Activation of DC cultures was independent of the lentiviral pseudotype, but dependent on cell entry and reverse transcription. In vivo transduced DC also displayed a mature phenotype, produced TNF-alpha, and stimulated naive CD8+ T cells. Lentiviral activation of DC was TLR-dependent as it was inhibited in TRIF/MyD88 knock out (-/-) DC. TLR3-/- or TLR7-/- DC were less activated, and reverse transcription was important for activation of TLR7-/- DC. Moreover, lentivirally transduced DC lacking TLR3 or TLR7 had an impaired capacity to induce antigen-specific CD8+ T cell responses. In conclusion, we demonstrated TLR-dependent DC activation by lentiviral vectors, explaining their immunogenicity. These data allow the rational development of strategies to manipulate the host's immune response to the transgene.