Dendritic cells and cytokines in immune rejection of cancer

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Cytokine & Growth Factor Reviews 19 (2008) 93–107 www.elsevier.com/locate/cytogfr

Dendritic cells and cytokines in immune rejection of cancer Maria Ferrantini *, Imerio Capone 1, Filippo Belardelli 2 Department of Cell Biology and Neurosciences, Istituto Superiore di Sanita`, Viale Regina Elena, 299, 00161 Rome, Italy Available online 3 December 2007

Abstract Dendritic cells (DCs) play a crucial role in linking innate and adaptive immunity and, thus, in the generation of a protective immune response against both infectious diseases and tumors. The ability of DCs to prime and expand an immune response is regulated by signals acting through soluble mediators, mainly cytokines and chemokines. Understanding how cytokines influence DC functions and orchestrate the interactions of DCs with other immune cells is strictly instrumental to the progress in cancer immunotherapy. Herein, we will illustrate how certain cytokines and immune stimulating molecules can induce and sustain the antitumor immune response by acting on DCs. We will also discuss these cytokine–DC interactions in the light of clinical results in cancer patients. # 2007 Elsevier Ltd. All rights reserved. Keywords: Dendritic cells; Cytokines; Immune potentiators; Cancer immunity; Immunotherapy

1. Introduction The concept of tumor recognition and elimination by the immune system was first advanced by Ehrlich in 1909 [1] and later formalized by Burnet [2] and Thomas [3] as the theory of immune surveillance. Since then, a large body of evidence has indicated the existence of both humoral and cell-mediated tumor-specific immune responses in cancer patients, both systemic and at the tumor site [4–6]. The recent progress in immunology has revealed the crucial role of dendritic cells (DCs) in the generation of a protective immune response against both infectious diseases and tumors [7], due to the unique ability of these professional antigen presenting cells (APCs) to prime naı¨ve T and B cells and, thus, to link innate and adaptive immunity [8]. DCs originate from pluripotent stem cells in the bone marrow, enter the blood stream and localize into almost all organs [9]. Based on the relative expression of a series of surface markers, different subsets of human DCs or DC * Corresponding author. Tel.: +39 064990 6087; fax: +39 064990 2140. E-mail addresses: [email protected] (M. Ferrantini), [email protected] (I. Capone), [email protected] (F. Belardelli). 1 Tel.: +39 064990 6089; fax: +39 064990 2140. 2 Tel.: +39 064990 6091; fax: +39 064990 3641. 1359-6101/$ – see front matter # 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.cytogfr.2007.10.003

precursors can be identified in peripheral blood, including a major CD1a+/CD11c+ and CD1a /CD11c+ population, expressing the CD13, CD33 and GM-CSF-receptor (referred as myeloid DCs), and a CD1a /CD11c population expressing high levels of CD123 (IL-3Ra), originally called lymphoid DCs and recently named as plasmacytoid DCs (pDCs), which represent the major type I interferon (IFN) producers upon virus challenge [10,11]. Human myeloid DCs comprise two major subsets expressing distinct phenotypes and functions: interstitial DCs, found in deep interstitial and epithelial tissues, and Langerhans cells, present in the epidermis [12]. DCs are found in tumor lesions in cancer patients, and, although the mechanisms by which they traffic to the tumor site have not been fully elucidated yet, several chemokines and cytokines have been implicated [13]. The mere presence of DCs in the tumor microenvironment is not necessarily conducive to the activation of antitumor immune responses, due to tumor-derived cytokines and growth factors that may suppress DC function or condition DCs to induce suppressive T cells [see the review article by Gottfried et al. in this issue]. It has been clearly demonstrated that the maturation and activation of DCs into powerful APCs by ‘‘danger’’ signals are essential requisites for an immune response to occur [14].

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Today, understanding how to condition DCs in vivo or manipulate them ex vivo can be considered as strictly instrumental to the progress in cancer immunotherapy. In fact, DCs can be considered as ideal tools for the development of therapeutic vaccines against cancer [7] and numerous clinical studies have been now performed to evaluate the safety and the possible efficacy of DC-based vaccines in cancer patients. However, DCs can differently orient the T cell responses, from thymic negative selection to the generation of effector and memory cells as well as to the induction of peripheral tolerance. The plasticity of DC functions is regulated by an ensemble of soluble factors (namely cytokines) that can either promote immunity or favor the induction of tolerance. Therefore, understanding how to exploit the recent knowledge on the interactions among DC subsets and between DCs and lymphocytes is pivotal for the identification of more effective and safe strategies for immunotherapy of cancer. In this review article, we provide an overview of the existing knowledge on the ability of cytokines and other immune stimulating agents to condition DCs in vivo or ex vivo towards the acquisition of functions promoting the generation of an antitumor adaptive immunity. Concerning the in vivo targeting of DCs, we will focus on cytokines and agents that have been more widely used in the clinical setting. We will also summarize and discuss the main results obtained in clinical trials based on the use of autologous DCbased vaccines in cancer patients.

2. DCs as in vivo targets of cytokines or immunopotentiators for induction of antitumor immune responses The understanding of the signals necessary to convert DCs into immunostimulatory APCs capable of priming

appropriate effector T cells has allowed identifying keymolecules suitable for the development of novel and effective adjuvants for cancer vaccines. Among them, certain cytokines and newly developed compounds have shown promise as potentially new adjuvants and are currently being tested in clinical studies. The scope of this section is to provide the reader with newly acquired knowledge on those immunopotentiators and cytokines recognized as strong DC activators in preclinical models and that have been used in clinical studies based on immunological interventions in cancer patients. Fig. 1 illustrates the main signals triggering DC activation in vivo and the cytokines promoting the generation of immune responses, as they will be discussed in the following sections. 2.1. Immunopotentiators and DCs The term immunopotentiators [15] defines the category of adjuvants exerting their effects directly on professional APCs (mainly the DCs) through specific receptors. An important impulse to the search of new immune potentiating adjuvants has been provided by the discovery of the socalled pathogen-associated molecular patterns (PAMPs), a family of evolutionary conserved structural elements shared by microbial pathogens, such as lypopolisaccharide (LPS) and CpG motifs, which can act as powerful activators of the immune system reviewed in ref. [16]. These compounds represent signatures of potentially noxious substances and in vertebrates are detected by families of receptors, collectively termed pattern-recognition receptors (PRRs) [16,17]. Importantly, these receptors are expressed on DCs that constantly screen the environment by using their PRRs as ‘‘sensors’’ for pathogens and translate the microbial stimulus into a biochemical signal inside the cell. This in turn leads to the transcriptional activation of proinflamma-

Fig. 1. Cytokines and immune stimulating signals triggering DC activation in vivo and promoting the induction of protective immune responses. Schematic representation of the cytokine cascade triggered by TLR agonists in pDCs ad myeloid DCs and involved in the generation and long-term maintenance of Th1 type immune responses, as described in details in the main text.

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tory cytokines and chemokines and up-regulation of costimulatory molecules, finally resulting in the activation of an adaptive immune response. The Toll-Like Receptor (TLR) family of PRRs is the best characterized and comprises 10 members identified in humans [16] Each TLR binds to distinct PAMPs: for example, TLR4 detects the LPS molecule specific for Gramnegative bacteria; TLR3 recognizes double-stranded RNA (Poly I:C); TLR7 can be triggered by the synthetic compounds imidazoquinolines; and the natural ligands for TLR9 are CpG-rich DNA motifs [16]. The TLRs are differentially expressed by distinct DC subsets and the trigger of the different TLRs may mediate distinct Thpolarised responses, or induce T regulatory pathways or CTL responses or antibody production. Thus, it is possible to selectively target functionally distinct DCs through the selection of specific TLR ligands. For example, the engagement of TLR7 and TLR9, selectively expressed by pDCs, induces a strong production of IFN-a, which subsequently stimulate Th1-type immune responses, but not of IL-12 (p70) [18]. Myeloid human DCs have been found to express all TLRs except TLR7 and TLR9 and to produce high levels of IL-12 upon TLR2- or TLR4-mediated activation [19]. Interestingly, in human myeloid DCs TLR3 and TLR4 have been shown to act in synergy with TLR7, TLR8 and TLR9 in the induction of an increased production of IL-12. This ‘‘combinatorial code’’ for optimal IL-12 production may allow myeloid DCs to efficiently polarize the immune response toward a Th1 type upon stimulation by pathogenor damage-associated molecular pattern molecules [20]. Interestingly, TLR3, TLR7 and TLR9 engagement by their relative ligands may also induce type I IFN production, which is well known to enhance cross-presentation by DCs and strong CTL activation reviewed in ref. [21]. In the following sections, we will focus on the immune stimulatory properties of those TLR synthetic agonists that have been more widely used in clinical trials for the treatment of cancer patients. The evidences of the potential correlations between the action of these molecules on DCs and the immunological and/or clinical response will be discussed. 2.1.1. CpG oligonucleotides Among the TLR agonists showing strong immunostimulatory effects on DC activation are the synthetic oligodeoxynucleotides (ODN) containing unmethylated CpG motifs (CpG ODN). CpG motifs are sequences typical of bacterial and viral genomic DNA but are uncommon and highly methylated in vertebrate genomes [22]. CpG ODN activate TLR9 that in humans is expressed in B cells and pDCs, whereas in mice B cells, monocytes, and apparently all DC subsets express TLR9 [16]. This difference between the human and mouse immune systems strongly impedes the extrapolation of the possible effects of TLR9 activation in humans from those observed in mice. Among the different

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families of CpG ODN with immunostimularory activities, the B-class CpG ODN (strong B cell stimulators but poor inducers of IFN-a in pDCs) are those more advanced in clinical development in oncology, being tested in phase II and phase III clinical trials as cancer vaccine adjuvants and in combination therapies reviewed in ref. [23]. CpG ODN activation of TLR9 leads to a remarkable induction of Th1 immune responses, resulting from the direct activation of B cells and pDCs as well as from the indirect activation of other immune cells and the consequent release of Th1 cytokines and chemokines [23]. The TLR9induced stimulation of B cells and pDCs can also promote the direct elimination of the tumor cells, through the secretion of IFN-a and the expression of TNF-related apoptosis-inducing ligand (TRAIL) [24], or the activation of killing by NK cells [25]. Interestingly, freshly isolated human NK cells have been shown to promote, in a CpGdependent manner, the release by pDCs of IFN-a, that in turn up-regulates NK-mediated killing [26]. The innate immunity mediated by TLR9-stimulated pDCs and B cells promotes the induction of antigen-specific adaptive immune responses, both humoral and CD4+ and CD8+ T cell-mediated [27–29]. Of note, pDCs stimulated with CpG ODN exhibit an increased capability of inducing antigen-specific memory CD8+ T cells and, in the case of Bclass CpG ODN, naı¨ve CD8+ T cells [30]. In a murine model of renal cell carcinoma, CpG ODN have been shown to potentiate the efficacy of a vaccine based on tumor antigenpulsed bone marrow-derived DCs in reducing tumor growth and preventing tumor implantation, as well as in conferring tumor-specific immunity [31]. Of particular relevance for the antitumor activities of TLR9 agonists are their effects on the functional properties of T regulatory (Treg) cells and on the role played by DCs in these interactions. The picture emerging from the studies in mouse models is rather complex, with evidences of TLR9induced DC capability of either overcoming Treg cell suppressive effects through the release of IL-6 [32], or to reverse Treg cell anergy by secreting IL-6 and IL-1 [33] and to increase Treg cell proliferation [33,34]. Similarly, human pDCs activated through TLR9 promote the generation of Tregs [35]. Finally, mouse DCs stimulated by different TLR ligands, including TLR9, have been demonstrated to play a crucial role in the in vitro differentiation of CD4+ T cells by producing IL-17, a key cytokine in tissue inflammation [36]. In mouse models, in vivo TLR9 activation by injection of CpG ODN has been shown to exert different effects in terms of impairment of Treg cell immune suppressive effects, possibly related to the route of administration, reviewed in ref. [23]. In humans, further investigation is needed to assess which route of administration of CpG ODN has to be preferred in order to effectively counteract the Treg cell suppressive effects, and also to evaluate the impact of Treg cell depletion or inactivation on the efficacy of CpG ODN treatment.

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The use of CpG ODN as vaccine adjuvants in humans has been prompted by the large body of evidence obtained in mouse models on the high efficiency of these compounds in inducing strong Th1 immune responses as well as antitumor responses, reviewed in ref. [23]. Moreover, the use of particular formulations can further improve the adjuvant activity of CpG ODN [37,38]. The adjuvant activity of CpG ODN has been demonstrated in clinical trials of vaccination of cancer patients. In melanoma patients vaccinated with a MART-1/Melan-A analog peptide, the addition of the TLR9 agonist CPG 7909 to incomplete Freund’s adjuvant (IFA) resulted in a 10-fold increase in the frequency of peripheral blood CD8+ T cells specific for the MART-1/Melan-A peptide [39]. Of note, these T cells had differentiated to effector cells; however, the peptide-specific lymphocytes in the tumor site, where high numbers of Treg cells were present, exhibited a less differentiated phenotype, and expressed lower levels of perforin, granzyme and inducible IFN-g [40]. A more recent study has shown that vaccination of cancer patients with recombinant NY-ESO-1 protein, Montanide ISA-51 (IFA), and CpG ODN 7909, resulted in the early induction of specific integrated CD4+ Th cells and antibody responses as well as of CD8+ T cells by in vivo cross-priming [41]. The therapeutic efficacy of TLR9 agonists is currently under evaluation in a number of clinical trials of vaccination [23]. Monotherapy with TLR9 agonists, administered without a vaccine, has been shown to activate NK cells and to induce a Th1 response in patients with B cell lymphomas, as well as to activate pDCs and, indirectly, myeloid DCs in melanoma patients [23]. Interestingly, a recent study reported the activation of both pDCs and myeloid DCs in the sentinel lymph node of early stage melanoma patients receiving intradermal CpG-B, together with the presence of a newly identified CD11chiCD123+CD83+TRAIL+ mature DC subset [42]. In current clinical trials using TLR9 agonists, much attention is focused on combination therapies, since the results on the therapeutic efficacy of CpG ODN as single agents have not been satisfactory so far. The potential advantage of combining CpG ODN administration with conventional or more novel cancer therapies, or with other strategies of cancer immunotherapy is suggested by a number of studies in mouse models [23]. Combining TLR9 activation with chemotherapy regimens has been shown to result in synergistic therapeutic effects [23,43]. This synergy may derive from the combination of the effects of chemotherapy, that in addition to deplete Treg cells also favors tumor antigen uptake by DCs through tumor destruction, with TLR9 activation of tumor antigen-loaded DCs and ensuing priming and/or boosting of tumor-specific effector Th1 immune responses. 2.1.2. Imidazoquinolines The need of relying on more potent and highly tolerable adjuvants prompted the development of new small-

molecules acting as immune potentiators. A family of such compounds is represented by the imidazoquinolines [44]. Among the imidazoquinolines, a synthetic agonist for TLR7, imiquimod, has been approved by the FDA for the topical treatment of genital warts, actinic keratosis, and basal cell carcinoma (BCC), and it is widely used also for other conditions [45]. Imiquimod acts through binding to TLR7 and, to a lesser extent, TLR8, both natural receptors for single-stranded RNA, leading to up-regulation of cytokines, mainly IFN-a as well as TNF-a, IL-1, IL-6, and monocyte chemoattractant proteins (MCPs) [45]. This cytokine-conditioned milieu promotes the recruitment and activation of DCs and macrophages and induction of Th1 responses within a few days of treatment [46], and leads to apoptosis of cancer cells and their substitution by a mononuclear cell infiltrate within 2 weeks [46,47–49]. The likely candidates for the initiation of the imiquimodinduced host defence reaction are pDCs and myeloid DCs, due to their prominent expression of TLR7 and TLR8, respectively. Indeed, the emergence of pDCs in the peritumoral tissue of imiquimod-treated human skin cancers has been demonstrated [50]. Other possible mechanisms for the antitumor activity of imiquimod are: an increased NK cell activity promoted locally by IFN-a [51], a reversal of Treg cell function [52], an immunostimulatory action via adenosine receptor signaling [53], proapoptotic effects on tumor cells, either direct [54] or indirect IFN-a-mediated [55], and an antiangiogenic activity [56,57]. The abundant presence of both CD4+ T cells and CD8+ T cells in the inflammatory infiltrate during imiquimod treatment-induced regression of cancerous lesions, [58–61], and the expression of molecules mediating the cytotoxic activity of CD8+ T cells [59,61] are strongly in support of an enhanced cytotoxic cell-mediated immune response being responsible for the imiquimod-induced clearance of cancer cells. A recent study attempted to characterize the imiquimod effects acting downstream of the induction of IFN-astimulated genes [62] through sequential gene profiling of basal cell carcinomas treated with imiquimod in a placebocontrolled study [63]. The results indicated a higher prevalence of IFN-g transcription versus IFN-a transcription, consistent with the parallel evidence of a predominant NK, CD8+ and CD4+ T cell activity. These findings suggest that imiquimod-activated pDCs trigger, through the production of IFN-a, the activation of other resident immune cells, such as NK and T cells, leading to the local release of high levels of IFN-g. The authors hypothesize that these effector mechanisms, by killing of cancer cells, favor the uptake of tumor antigens by professional APCs, mainly DCs, and thus the priming of naive T cells in draining lymph nodes. If proven, this mechanism of action of imiquimod and possibly of other TLR7 agonists would imply the capability of these compounds to overcome the limitation often observed with cancer vaccines, i.e. successful elicitation of tumor antigenspecific T cells but ineffective activation of T cells at the tumor site.

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Among the effector cells elicited by TLR7 agonists, both myeloid DCs and pDCs have been described by a very recent immunohistochemistry analysis of the inflammatory infiltrate of BCC lesions treated with topical imiquimod [64]. Interestingly, peritumoral myeloid DCs expressed perforin and granzyme B, whereas infiltrating pDCs expressed TRAIL. Moreover, the myeloid DCs coexpressed TNF-a and iNOS, two proinflammatory mediators with antitumor activity [65,66]. These findings lead to envisage a major role of DCs not only in the initiation but also in the effector phase of the immune response. In mouse tumor models, imiquimod has been used as an adjuvant of cancer vaccines and several reports indicate its ability to enhance antitumor immune responses as well as the therapeutic efficacy of vaccination [67,68]. Among the mechanisms responsible for the adjuvant activity of imiquimod administered with cancer vaccines are a DCmediated bypassing of peripheral tolerance Craft et al. [67], and the enhancement of DC survival and trafficking and of the priming of tumor-specific CD8+ T cells [68]. The interesting possibility of using imiquimod as a topical adjuvant has been indicated by an approach alternative to in vitro maturation of DCs, consisting of in vivo DC activation by injecting immature DCs into a cutaneous site pre-treated with imiquimod. Of note, this approach resulted in enhanced DC migration [69]. In melanoma patients treated with Flt3 ligand and receiving vaccination with influenza (Flu), Melan-A (Mel), tyrosinase (Tyr), and NY-ESO-1 peptides, the topical application of imiquimod at the vaccine sites resulted in the development of a specific CD8+ T-cell response to NY-ESO-1 peptide in the majority of patients (5/8) [70]. 2.2. Cytokines and DCs As highlighted in the previous sections, the cross talk between innate and adaptive response is mediated by cytokines, and DCs are the major players involved in this process [71]. In particular, certain cytokines are produced rapidly in response to pathogens or ‘‘danger signals’’ and can profoundly affect the function of DCs, by inducing differentiation, activation, maturation and migration of these types of cells. It has become increasingly evident that adjuvants generally act through the induction of cytokines, which are the key mediators of the immune response. This fact has raised the question of whether the direct use of certain selected cytokines as natural adjuvants could be of some advantage with respect to the conventional adjuvants. In this session, we focus on two cytokines known to play an important role in linking innate and adaptive immunity by acting on DCs, namely GM-CSF and IFN-a. Our choice is motivated by the wide clinical use of these cytokines in cancer patients, either as cancer vaccine adjuvants, mostly in the case of GM-CSF, or as antitumor cytokine, in the case of IFN-a. The available data on the activity of these cytokines

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in cancer patients offer the possibility of interpreting or reinterpreting the clinical response to GM-CSF as adjuvant of cancer vaccines and to IFN-a in the light of the recent knowledge on their immunomodulatory effects that are largely mediated by their acting on DCs. It should be kept into consideration, however, that some of the effects induced by these cytokines may be due to the induction of other cytokines. For example, part of the IFN-ainduced effects on differentiation/activation of DCs and expansion/survival of memory CD8+ T cells may be mediated by IL-15, which is produced by DCs in response to IFN-a [72] and plays a pivotal role in the activation of NK cells and in the maintenance of long-lasting, high-avidity CD8+ memory T cells [73]. Of note, IL-15 has been recently shown to exert biologic effects on the DCs themselves, affecting DC functions important for the induction of antitumor immunity, through the enhancement of the DC capability to polarize the differentiation of naı¨ve T cells into Th1 type cells in the mouse [74], the restoration of the MHC class I antigen-processing machinery in human DCs inhibited by tumor-derived gangliosides [75], and the augmentation of human DC efficiency in priming melanoma-specific CD8+ T cells endowed with superior effector functions [76]. The best known DC-derived soluble factor mediating the effects of cytokines and other immune stimulating agents is IL-12, a key regulator of the induction of adaptive Th1 type immunity [77]. Recently, new knowledge on the immune regulatory activities of IL-12 has been acquired, indicating this cytokine as a critical third signal for CD8+ T cell differentiation [78,79], and as an important factor that, by promoting the reactivation and survival of memory CD4+ T cells [80], can contribute to the repolarization of dysfunctional antitumor Th2 CD4+ T cells into Th1 cells [81]. The use of IL-12 for the treatment of cancer patients has been prompted by the promising results obtained in a number of animal tumor models on the strong antiangiogenic and immune-mediated antitumor activities of IL-12 (reviewed in [82]). IL-12 has been used for the treatment of patients with advanced solid tumors and hematologic malignancies, either as a single agent or in combination with other therapies (reviewed in [77]). However, the clinical use of IL-12 has been restricted by severe toxicity and, overall, has been associated with a limited efficacy [77], for reasons that have not been completely understood. Combination therapies based on the use of IL-12 can be envisaged that might result in a better therapeutic benefit for cancer patients [77]. 2.2.1. GM-CSF as adjuvant of cancer vaccines The clinical use of GM-CSF as an adjuvant of cancer vaccines against a variety of human neoplastic diseases has been based on the rationale of exploiting the immune stimulatory effects of this cytokine, largely documented in animal models [83]. In particular, the capability of GM-CSF to induce the local recruitment and maturation of DCs [84] has been considered to play a key role in promoting the

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presentation of tumor-associated antigens (TAAs) to T lymphocytes in the lymph nodes as well as the activation of innate immunity effector cells [83]. The results of the clinical trials in which GM-CSF has been used as an adjuvant of cancer vaccines, either released by gene-transduced tumor cells or co-administered as recombinant protein with the vaccine, are illustrated and discussed in a recent review by Parmiani et al. [85]. The authors point out the heterogeneity of the results in terms of induction of vaccine-specific immune response and of clinical response, with some studies supporting a positive effect of GM-CSF in the generation of an immune response, and others reporting no effect or a suppressive effect. Based on their detailed analysis, the authors conclude that repeated administration of GM-CSF at relatively low doses 40–80 mg may increase the vaccine-induced immune response, whereas regimens based on higher dosages (100–500 mg) often have resulted in suppression of the immune response. This dose-dependent dichotomy in the mode of action of GM-CSF may be explained by the ability of this cytokine to activate and expand myeloid suppressor cells (MSC) that inhibit T cell functions, as clearly demonstrated in mouse models [86]. These studies suggest that sustained systemic levels of GM-CSF promote bone marrow mobilization of immature Gr1+CD11b+ myeloid cells [87] that mediate immune suppression by inhibiting T cell function through the concomitant production of arginase 1 and a shift from nitric oxide (NO) to urea/ornithine production [86]. These findings may explain the immune suppressive effects observed in some clinical trials based on the use of GMCSF as adjuvant of cancer vaccines. In cancer patients, the presence and expansion of MSC, likely the result of tumor-derived myeloid growth factors such as GM-CSF, has been clearly demonstrated [88]. In the case of patients with head and neck carcinoma these cells have been linked to the ability of tumor cells to release GMCSF [89,90]. Human MSC apparently differ, depending on the tumor histotype with which they have been found associated, in terms of phenotypic and functional properties as well as of mechanisms of immune suppression [88]. However, they could represent differentiation-arrested myeloid DC precursors whose expansion would have the dual negative effect of impairing the generation of fully functional DCs and inhibiting T cell functions, through the production of NO [91,92]. In conclusion, although further clinical studies based on a more homogeneous and standardized use of GM-CSF would be needed in order to definitely assess its potential as adjuvant of cancer vaccines, the ensemble of data available at present on its immunomodulatory effects should force to exercise great caution in the administration of this cytokine to cancer patients. 2.2.2. IFN-a IFN-a are cytokines belonging to type I IFNs and exerting a variety of biological effects including those on

viral replication and antitumor activity (reviewed in [93,94]). IFN-a represent the cytokines exhibiting the longest record of use in clinical oncology for the treatment of more than 14 types of cancer, including some hematological malignancies (hairy cell leukemia, chronic myeloid leukemia, some B- and T-cell lymphomas) and certain solid tumors, such as melanoma, renal carcinoma and Kaposi’s sarcoma. Even though today some new anticancer drugs have somehow replaced IFN-a in the treatment of certain hematological malignancies (i.e., hairy cell leukaemia and chronic myeloid leukemia), this cytokine is still widely used in the treatment of patients with specific types of tumor, such as metastatic melanoma, and viral diseases (hepatitis C). However, in spite of many years of intense work in animal tumor models and of considerable experience in the clinical use of IFN-a, the importance of the different mechanisms of action underlying the response in patients is still matter of debate. In addition to the direct effects on tumor cells, considered for a long time the major mechanisms of the clinical response in IFN-treated patients [94], IFN-a exert several effects on host immune cells that can play a central role in the overall antitumor response and that have led to the recognition of the importance of these cytokines in tumor immunity [95,96,97]. Of interest, the results of clinical studies reveal new immune correlates of clinical response which might be predictive of antitumor efficacy [98–100]. Today, new attention is given to IFN-a as an important factor linking innate and adaptive immunity, based on the evidence of the importance of type I IFNs in the differentiation of the Th1 subset, as well as in the generation and activity of cytotoxic T-lymphocytes (CTL) (reviewed in [101]. In particular, type I IFNs are important for the in vivo proliferation and expansion and long-term survival of CD8+ T cells in response to specific antigens [102], and for the adjuvant activity on T cells induced by CpG DNA administration [103]. The renewed interest in IFN-a as a ‘‘bridge system’’ linking innate and adaptive immunity stemmed from the identification of ‘‘natural IFN-producing cells’’, also defined as plasmacytoid DCs (pDCs) [see the review article by Fitzgerald-Bocarsly et al., in this issue]. In recent years, a growing body of evidence has indicated that IFN-a can exert important effects on the differentiation and function of DCs and that such effects may play major roles in the induction of IFN-induced antitumor immunity. Thus, several groups, including our laboratory, could show that IFN-a acted as an important signal for differentiation and activation of DCs [72,104–106]. In particular, IFN-a promotes the rapid differentiation of GM-CSF-treated human monocytes into highly active DCs, exhibiting the phenotype of partially mature DCs, as revealed by the expression of DC markers and migratory response to chemokines. [72]. Notably, these DCs were endowed with potent functional activities, not only in vitro but also in vivo, as evaluated by the strong capability of these cells to induce the generation of a primary human antibody response and

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CTL expansion both in vitro and after injection into SCID mice reconstituted with human peripheral blood lymphocytes (Hu-PBL-SCID mice) [72,107,108]. IFN–DCs proved to be superior with respect to the reference monocytederived DCs generated in the presence of IL-4 and GM-CSF in inducing a CTL response [107–109] and in vivo crosspriming of CD8+ T cells against exogenous viral antigens [110]. All this points out the existence of some sort of ‘‘natural alliance’’ between IFN-a and monocytes/DCs, which might be instrumental for the prompt generation of a protective immune response against pathogens as well as against cancer cells. In particular, the exposure of monocytes to IFN-a can represent the early mechanism involved in the maturation/induction of DCs in response to virus infection and possibly to other invading pathogens or tumors. A special interest on the biological significance of the IFN-a-DC interactions in the regulation of both normal and pathologic immune responses has stemmed from studies carried out by Banchereau and Pascual [111]. These authors described the phenotype and functions of DCs rapidly generated from monocytes exposed to IFN-a-containing sera from patients with systemic lupus erythematosus. Of interest, these studies also led to the suggestion of a possible role of IFN–DC interactions in the pathogenesis of autoimmune diseases (reviewed in [111]). On the whole, the results on IFN–DC interactions support the concept that IFN-a represents a powerful natural adjuvant for the connection between innate and adaptive immunity by acting on DC differentiation/activation. This knowledge can suggest new rationales for using this cytokine as an adjuvant in clinical studies: (i) in vivo, by injecting the cytokine together with a cancer vaccine with the aim of targeting patient’s DCs/DC precursors and inducing their differentiation/activation; (ii) in vitro to generate IFN–DCs from patient’s monocytes; these IFN–DCs would be then loaded with the relevant tumor antigens and reinfused into the patient. A compelling evidence for the importance of the immunomodulatory effects of IFN-a in its therapeutic activity against human malignancies stems from studies on chronic myeloid leukemia (CML). In particular, the demonstration of a strong correlation between the clinical response of CML patients to IFN-a therapy and the presence of T cells specific for an epitope targeted by CML-specific T cells [99], as well as of antibodies against CML-associated antigens [98] suggests that IFN-a might induce remission by promoting the expansion of autologous leukemia-specific effector T cells and/or B cells. Other studies suggest that a possible mechanism by which IFN-a stimulates an antileukemia immune response in CML patients is the promotion of the differentiation of highly active DCs [112–114]. The results of these studies together with clinical data suggesting that the addition of GM-CSF to IFN-a therapy can significantly improve the cytogenetic response in some patients [115] strongly support the hypothesis that the combined administration of GM-CSF and IFN-a to CML

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patients can promote the generation of highly active DCs from malignant cells. Of special interest, in a recent study published by Gogas et al. in melanoma patients treated with the high dose IFN-a regimen [100], a striking correlation between clinical response to IFN-a and autoimmune manifestations was observed. The results of this study are somehow consistent with the hypothesis of a possible role of IFN–DCs in the pathogenesis of autoimmune responses [111] and may lead to new perspectives for identifying categories of patients responding to the IFN-a therapy [116]. Likewise, this study further supports the interest of using IFN-a in association with cancer vaccines. In this regard, we have recently carried out a pilot phase I–II trial to determine the effects of IFN-a, administered as an adjuvant of MelanA/MART-1:26–35(27L) and gp100:209–217(210M) peptides, on immune responses in stage IV melanoma patients [117]. In five out of the seven evaluable patients, a consistent enhancement of CD8+ T cells recognizing modified and native MART-1 and gp100 peptides and MART-1+gp100+ melanoma cells was observed. Moreover, vaccination induced a raise in CD8+ T-cell binding to HLA tetramers containing the relevant peptides and an increased frequency of CD45RA+CCR7 (terminally differentiated effectors) and CD45RA CCR7 (effector memory) cells. In all patients, treatment augmented significantly the percentage of CD14+ monocytes, and particularly of the CD14+CD16+ cell fraction. Notably, post-vaccination monocytes from two out of the three patients showing stable disease or long disease-free survival showed an enhanced APC function and capability to secrete IP10/CXCL10 when tested in MLR assays, associated to a boost of antigen and melanomaspecific CD8+ T cells. The studies reviewed above strongly support the rationale for using IFN-a as immunopotentiators for the generation of more effective cancer vaccines by acting on DCs and/or by enhancing T-cell functions. The results of pilot clinical studies may support these two hypotheses [117,118]. To this regard, it should be taken into consideration that IFN-a exerts its effects both on the host’s immune system and on tumor cells, possibly setting the scene for a cooperative action in the generation of longlasting control of tumor growth. For example, IFN-a can prime the tumor cells for apoptosis induction by different stimuli (reviewed in [101]). Notably, human DCs acquire and efficiently present antigens derived from apoptotic cells, resulting in cross-priming and CTL generation, and IFN–DCs appear to be particularly effective in taking up apoptotic bodies and inducing cross-priming of CD8+ T cells against defined antigens (our unpublished results). Thus, the fact that IFN-a can favor apoptosis and that the same cytokine can induce the differentiation/activity of DCs endowed with a special capability of apoptotic body uptake and CD8+ T-cell cross-priming makes sense in explaining the correlation recently observed between clinical response and manifestation of autoimmune reactions in melanoma patients subjected to the high dose

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3. Cytokines in the ex vivo generation of DCs for the development of therapeutic vaccines

Fig. 2. Hypothetical scenario of events triggered by IFN-a leading to antitumor immunity and associated autoimmunity. IFN-a, either exogenously administered as a therapeutic agent or endogenously released by host’s cells, including pDCs, can exert the dual role of favoring tumor cell apoptosis [101] and inducing the differentiation of myeloid DCs highly active in taking up apoptotic bodies and in cross-priming CD8+ T cells against defined antigens [110]. Additional players in this scenario might be represented by pDCs, recently shown to mediate cross presentation of exogenous antigens acquired from vaccinal lipopeptides or from apoptotic virus-infected cells [119]. It might be also envisaged that pDCs respond to the signals conveyed by the apopotic tumor cells by releasing IFN-a, thus boosting the system. The final outcome of this concerted action would be the induction of potent Th1 type immune responses potentially directed against all the antigens expressed by the apoptotic tumor cells, including selfantigens, leading to the coupled development of antitumor immunity and autoimmunity.

IFN-a regimen [100] (Fig. 2). Of note, a very recent study has provided the first evidence of antigen cross presentation by human pDCs [119]. Interestingly, this newly recognized property of pDCs was greatly enhanced upon influenza virus infection [119], strongly suggesting the involvement of type I IFNs. These findings, together with the increasing evidence of the role of pDCs and type IFNs in the pathogenesis of autoimmune diseases in humans [120,121], put forward the challenging hypothesis of pDC involvement in the IFN-induced immune responses leading to antitumor immunity and associated autoimmunity.

The DC-based immunotherapy approach most commonly used so far is based on the ex vivo generation of DCs, their loading with tumor antigens and re-injection into the patient for stimulating cell-mediated immunity, taking advantage of the ability of DCs to migrate to the T-cell areas of lymphoid organs. Cytokines have represented invaluable tools for the ex vivo differentiation and activation of DCs from their precursors, and thus for the development of DC-based therapeutic cancer vaccines. The first ‘‘proof of concept’’ clinical trials using DCbased therapeutic cancer vaccines have been published more than 10 years ago, and the knowledge acquired through these and subsequent studies has provided important insights for the development of DC-based vaccines capable of inducing immune responses and in some cases clinical responses in cancer patients. Nevertheless, there is no consensus yet on the methods to be used for the preparation of DCs, as well as on the methods for antigen loading for generating fully competent DCs for vaccine-based clinical studies. This stems by the fact that DCs exist as diverse populations and at different stage of maturation, with unique functional capacities and mainly classified as ‘‘myeloid’’ or ‘‘plasmacytoid’’ DCs. Different DC subsets, stimulated by various activation signals, can lead to different T cell polarizations (i.e. Th-1 or Th-2 cells) [122]. This is a crucial factor in vaccination against cancer, since Th-1 immunity is considered as advantageous, while Th-2 immunity can be deleterious. Moreover, DCs can induce immune tolerance, both because of antigen presentation in the absence of appropriate co-stimulation in the case of immature DCs, and by stimulating the expansion of naturally occurring CD4+CD25+ Treg cells [123]. In this section, we will briefly review the classical and recently developed protocols for in vitro generation of DCs and their use to prepare DC-based cancer vaccines. The main outcomes of recent DC-based clinical trials in cancer patients will also be illustrated. 3.1. Methods for ex vivo generation of DCs The methods most frequently applied for obtaining DCs are based on the ex vivo generation of DCs from precursors. Fig. 3 illustrates three methods of DC generation from progenitor cells. The first one is based on the use CD34+ blood or bone-marrow-derived precursors, expanded in the presence of a cocktail of cytokines, including stem cell factor, Flt3L, IL-3, and IL-6, and differentiated/maturated with GM-CSF, IL-4, and TNF-a (reviewed in [124,125]). CD34+-derived DCs contain a mixture of interstitial DCs and DCs of the Langerhans type, and are CD11c+ CD1a+ CD14 HLA-DR+. Although CD34+-derived DCs are capable of inducing tumor-antigen-specific immune responses [126], including the expansion of CTLs recogniz-

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Fig. 3. Main methods for generation of human DCs for the preparation of DC-based therapeutic cancer vaccines from precursor cells cultured in the presence of different cytokines. (A) CD34+ cells are first cultured for 5–7 days in the presence of a cytokine cocktail (SCF, Flt3-L, IL-3) for generating and expanding DC precursors, which are then incubated for an additional 5–7 days in the presence of different cytokines, such as GM-CSF, IL-4, and TNF-a, to generate different types of functionally active DCs, i.e. interstitial DCs and DCs of the Langerhans type [123,124]. (B) Exposure of monocytes to IL-4 (alternatively IL-13) and GM-CSF drives their differentiation into immature DCs [127,128]. This phenotype is lost upon cytokine deprivation or diverted upon exposure to different cytokines. Immature DCs are weak APCs, due to low/moderate surface expression of costimulatory molecules. Their activation/terminal maturation can be promoted by the addition of stimuli such as bacterial or viral components or products, recombinant soluble CD40L, macrophage-conditioned medium (MCM), and cytokines, including IL-1b and TNF-a, which induce the expression of the maturation marker CD83 and a marked upregulation of costimulatory molecules [129–131]. (C) Exposure of monocytes to type I IFNs and GM-CSF rapidly induces their differentiation into partially mature DCs (IFN–DCs) expressing higher levels of costimulatory molecules and low to moderate levels of CD83 [72,134,135].

ing tumor cells expressing target antigens [127], very few clinical trials have been conducted with CD34+-derived DCs. Thus, further clinical investigation is required in order to compare their effectiveness with respect to other types of DCs. The most common method for ex vivo generation of DCs employs purified CD14+ monocytes cultured in the presence of a variety of cytokine cocktails (the so-called Mo-DCs). The biology of Mo-DCs has extensively studied by several groups and most clinical studies have been carried out by using Mo-DCs. Large numbers of immature Mo-DCs can be generated from CD14+ monocytes exposed to GM-CSF and IL-4 [128] or IL-13 [129]. These DCs are weak antigen presenting cells, due to low/moderate surface expression of co-stimulatory molecules. Nevertheless, immature DCs have largely been employed in the earlier clinical trials, and only some recent studies, comparing the immunogenicity of immature versus mature DCs, have revealed that maturation of DCs is an essential step for inducing adequate immune responses in cancer patients [130,131]. Immature Mo-DCs can be differentiated into mature Mo-DCs by exposure to different maturation stimuli, such as Toll-like

receptor (TLR) ligands (such as LPS, poly-I:C, etc.), proinflammatory cytokines (such as TNF-a), CD40L or the socalled ‘‘gold standard’’ (IL-1b, TNFa, IL-6 and PGE2), also known as monocyte-conditioned medium (MCM) mimic or cytokine cocktail [132]. Mo-DCs matured in the presence of this cytokine cocktail are those most commonly used to treat cancer patients in clinical trials. However, a recent phase III clinical study [133] showed that vaccination of melanoma patients with cytokine cocktail-maturated DCs does not provide a benefit over the standard dacarbazine (DTIC) treatment. Even though the two-step protocol described above (i.e., DC differentiation, in the presence of IL-4 or IL-13, and DC activation, after exposure to maturation factors) has allowed to perform the vast majority of clinical studies published so far and to define mechanisms of DC activation, it might be argued that DCs generated after several days of in vitro exposure of monocytes to high levels of cytokines such as IL-4 or IL-13 hardly reflect the possible scenario of a physiological exposure of monocytes to cytokines induced in vivo following an antigen stimulation. Rapid single-step DCs generation protocols have been developed, allowing the

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production of DCs capable of stimulating T cell responses in vitro as effectively as DCs generated by standard protocols. Our group has developed a new protocol allowing the differentiation of CD14+ monocytes into highly active partially mature DCs in 3 days of culture in the presence of type I IFN and GM-CSF [72,134]. Interestingly, IFN–DCs produced significant IL-15 amounts in culture supernatant without any further stimulus, expressed CCR7 and showed a migratory response to CCL19 [134]. The IFN–DCs proved to be highly efficient in inducing, both in vitro and in vivo (in Hu-PBL-SCID mice), the priming of human antibody and cellular responses [72,107–109], as well as the crosspriming of CD8+ T cells against exogenous viral antigens [110]. IFN-b has been also used to generate DCs, exhibiting similar properties of IFN–DCs that have been shown to induce CD8+ T cell responses against a tumor-specific peptide in melanoma patients [135]. In a recently published clinical trial [136], DCs generated from monocytes in a 2-day culture in the presence of IFN-g and LPS have been used. Interestingly, these DCs are characterized by a mature phenotype and transiently secrete IL-12; however they are not exhausted, being able to secrete more IL-12 in response to CD40 stimulation. 3.2. Clinical trials with DC-based vaccines Over the last years, a remarkable interest has been focused on the attempts to use DCs in the development of therapeutic vaccines against cancer (reviewed in [137]). Table 1, based on a catalog of clinical trials compiled by Hart [138], illustrates the results of clinical trials based on DC vaccination against those tumors representing the most relevant targets for clinical experimentation. At this stage, a considerable number of patients, at least in the case of melanoma and prostate cancer patients, has been treated with DC-based vaccines, although different antigens, methods for DC generation and injection, as well as antigen loading approaches have been used, thus rendering somehow difficult the interpretation of the results. Nevertheless, as for the solid tumors reported in Table 1 it is evident that, a fraction of patients, ranging from about 30 to 50%, showed vaccine-specific immune responses, and only a small portion of them (from approximately 6 to 10%) experienced a clinical response. In addition, only in a few cases (see ref.

[137] for references) a correlation between immune and clinical responses has been observed, thus leaving unsolved the question whether the observed responses were actually caused by vaccination or rather they reflected patients’ immunocompetence status. Recently, a randomized phase III clinical trial in stage IV melanoma patients failed to demonstrate the superiority of DC-based vaccination over the dacarbazine (DTIC) chemotherapy [133]. In fact, the objective response in the DTIC (55 patients) and DC (53 patients) treatment arms were 5.5% and 3.8%, respectively. This difference, although statistically not significant, indicates a trend of worse clinical response in the DC-treated patients. On the other hand, a smaller, one arm clinical trial in metastatic melanoma patients vaccinated with DCs pulsed with irradiated autologous tumor cells showed very interesting results. Of 21 patients enrolled, 18 exhibited a median survival of 3 years, ranging from 2 to 5 years [139]. In particular, six of these patients, all of whom had not evidence of disease at enrollment but had experienced extensive metastatic disease or repeated recurrences treated with surgery and multiple therapies, remained disease-free for an interval of 24–58 months after the completion of the therapy. This time period appears highly significant if compared to the longest progression-free interval, i.e. 14 months, observed in one of those patients between two consecutive therapies prior to vaccination. The long progression-free survival of these patients is unusual and rarely observed in DC vaccination trials performed so far. The same authors conclude that it is unlikely that the vaccine alone produced the observed clinical benefit, considering that all the patients underwent more than one type of treatment before entering the study. Another similar striking result was observed in HIV-infected individuals, vaccinated with DCs pulsed with chemically inactivated autologous virus, in whom viremia lowered close to undetectable levels following DC vaccination [140]. Do these trials share some common features? Indeed, both trials used DC maturated in the absence of PGE2 and loaded with ‘‘endogenous’’ unprocessed antigens. The inclusion of PGE2 in DC maturation cocktails has been criticized [137]. Indeed, PGE2 endows DCs with high capability to migrate [141,142], but also renders them resistant to in vivo licensing by costimulatory molecules such as CD40. In fact, these DCs fail to induce IL-12 and produce immune

Table 1 Summary of the results of DC-based clinical studies in cancer patients Tumor type

No. of patients

Immune response (%)

Clinical response

Clinical response rate (%)

Melanoma Prostate cancer RCC CRC

573 530 185 121

218/412 (52.9) 182/324 (56.1) 63/137 (45.9) 26/92 (28.2)

61 31 16 0

10.6 5.8 8.6 0

Data are based on the catalogue of clinical trials with DCs compiled by Hart [138]. Patients are those considered assessable, and immune response is reported by the ratio between the number of patients showing immunity elicited by vaccination (detected by any kind of immune assay such as ELISPOT, Tetramer staining, DTH) and the number of patients actually tested. Clinical response and clinical response rate are calculated on the basis of assessable patients showing either partial response or complete response.

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suppressive factors such as IL-10 [143]. In addition, PGE2 is not capable to trigger an effective functional maturation of DCs [144], and cytokine cocktail-matured DCs have been shown to promote the expansion of Treg cells expressing FOXP3 even more efficiently than immature DCs [145].

4. Final remarks The recent knowledge on the biology of DCs has progressively revealed the complexity of the interactions among DC subsets and of DCs with other cells of the immune system as well as with tumor cells. The key role played by cytokines and chemokines in establishing, orienting and fine-tuning these interactions has become increasingly evident. DCs both produce and respond to soluble factors, which, depending on their quality, quantity and combinations, differently affect the functional outcome of DC interplay with other host’s cells. Thus, cytokines are the signs of ‘‘codes’’ used by DCs for communicating within the immune system. Deciphering these codes is pivotal for identifying how cytokines and cytokine-conditioned DCs can be used for inducing and sustaining effective immune responses against cancer. The unravelling of the mechanisms triggered by cytokines acting on DCs has already led to the clinical development of new adjuvants as well as of DC-based therapeutic cancer vaccines currently used in clinical trials. There are many questions that still need to be addressed in order to optimally exploiting the adjuvant activity of cytokines for enhancing the therapeutic efficacy of DCbased cancer vaccines. For instance, which cytokines or cytokine combinations should be preferred for the in vivo or ex vivo differentiation and activation of DCs? We still need to improve our knowledge on how to use those DC-targeting cytokines with a long record of clinical use, such as IFN-a and GM-CSF. Results from relevant clinical studies, already obtained or expected to be available in the near future, will provide the basis for further investigation. If we have now good chances of developing novel and more effective strategies for acting on DCs in order enhance vaccine efficacy, great attention and major expectations are focused on DC-based therapeutic vaccines to be used in patients with cancer. In the light of the complex biology of DCs and in the view of an expanding clinical use of DCs for immunotherapy, a requirement for successful clinical immune intervention is the definition of standardized procedures for both DC generation and quality assessment, with a particular emphasis on DC capability to induce helper and cytotoxic T cells with a high avidity for tumor antigens but few Treg cells [7]. The identification of the ideal injection route for DC-based cancer vaccines also needs systematic comparative clinical trials. To this regard, conditioning of the injection site with certain cytokines in order to promote migration of the injected DCs to the lymph node might represent a promising approach.

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However, in spite of the several still unclear aspects regarding the optimal modalities for preparing and using DC-based cancer vaccines, the results of the clinical trials realized so far indicate unequivocally the efficient induction of tumor-specific T cell immune responses in patients with advanced cancer (Table 1). This starting point paves the way for further clinical research combining DC-based vaccination with other conventional or novel therapeutic interventions in cancer patients.

Conflict of interest statement The authors declare that they have no competing financial interest.

Acknowledgements We are grateful to Mrs. Alessandra Mariani and Mrs. Cinzia Gasparrini for excellent secretarial assistance. Work in the authors’ laboratory was supported in part by grants provided by the European Union (6th Framework Programme, LSHB-CT-2003-503583) and AIRC.

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starting working at the Institute de Recherches Scientifiques sur le Cancer (IRSC), Villejuif (Paris) in the laboratory of Dr Gresser, the pioneer of the main discoveries on the interferon system. Then he worked at the Deptartment of Immunology of The Scripps Research Institute (La Jolla, CA). In 1983 he became Head of the Section of Biology and Genetics of Animal Viruses of the Deptartment of Virology at the Istituto Superiore di Sanita` (ISS) in Rome. His main areas of research include: (i) role of interferons and other cytokines in the pathogenesis and control of viral infections and in the immune response against tumors; (ii) cancer vaccines; (iii) biology of dendritic cells and development of immunotherapy strategies for infectious diseases and cancer. His research has built the basis for bringing together the results obtained in animal models to clinical applications, especially in the field of innovative immunotherapy and combination therapies for cancer. Since 2006 FB serves as Director of the Department of Cell Biology and Neurosciences at ISS, and as Scientific Director of the ISS GMP facility FaBioCell (Cellular and Biological Drugs), which is dedicated to the preparation of cellular products for clinical use. Maria Ferrantini Maria Ferrantini began her scientific career at the Istituto Superiore di Sanita` (ISS), by working on her thesis in the laboratory of Virology. She started studying the effects of type I IFN gene transfer in different types of mouse and human tumors in animal models. Her main research interests are linked to the development of antitumor gene therapy and immunotherapy approaches. She is at present actively involved in the study and design of innovative antitumor immunotherapy strategies based on the use of dendritic cells. Since 2006 she serves as Head of the Section of Experimental Immunotherapy of the Department of Cell Biology and Neurosciences at ISS, and is also responsible of the Research and Development unit of FaBioCell. Imerio Capone Imerio Capone received his PhD at the University of Rome ‘‘La Sapienza’’ in 1986. He has been working for many years at the Department of Genetics and Molecular Biology of the University ‘‘ La Sapienza’’, focusing his scientific activity on the control mechanisms of eukaryotic gene expression. In 1999 he moved to the Laboratory of Virology at the Istituto Superiore di Sanita` to provide his expertise in molecular biology applied to the development of new antiviral vaccination strategies. He is currently interested in developing new strategies for cancer therapy, with particular focus on dendritic cellbased and combination therapies. At present, Imerio Capone works at the Department of Cell Biology and Neurosciences, where he is involved in the design of clinical trials of cancer immunotherapy.