Immune modulation by genetic modification of dendritic cells with lentiviral vectors

Virus Research 176 (2013) 1–15

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Review

Immune modulation by genetic modification of dendritic cells with lentiviral vectors Therese Liechtenstein a,b , Noemi Perez-Janices a,c , Christopher Bricogne a , Alessio Lanna a , Inès Dufait g , Cleo Goyvaerts g , Roberta Laranga a , Antonella Padella a , Frederick Arce a , Mehdi Baratchian a , Natalia Ramirez d , Natalia Lopez e , Grazyna Kochan f , Idoia Blanco-Luquin c , David Guerrero-Setas c , Karine Breckpot g , David Escors a,b,∗ a

Division of Infection and Immunity, Rayne Institute, University College London, London, UK Immunomodulation Group, Navarrabiomed, Miguel Servet Foundation, Pamplona, Navarra, Spain c Epigenetics Group, Navarrabiomed, Miguel Servet Foundation, Pamplona, Navarra, Spain d Oncohematology Department, Navarrabiomed, Miguel Servet Foundation, Pamplona, Navarra, Spain e Cardiovascular Department, Navarrabiomed, Miguel Servet Foundation, Pamplona, Navarra, Spain f Structural Genomics Consortium, Oxford University, Oxford, UK g Vrije Universiteit Brussels, Jette, Brussels, Belgium b

a r t i c l e

i n f o

Article history: Received 18 April 2013 Received in revised form 13 May 2013 Accepted 14 May 2013 Available online 28 May 2013 Keywords: Dendritic cell PD-L1 PD-1 Costimulation Cancer TCR

a b s t r a c t Our work over the past eight years has focused on the use of HIV-1 lentiviral vectors (lentivectors) for the genetic modification of dendritic cells (DCs) to control their functions in immune modulation. DCs are key professional antigen presenting cells which regulate the activity of most effector immune cells, including T, B and NK cells. Their genetic modification provides the means for the development of targeted therapies towards cancer and autoimmune disease. We have been modulating with lentivectors the activity of intracellular signalling pathways and co-stimulation during antigen presentation to T cells, to fine-tune the type and strength of the immune response. In the course of our research, we have found unexpected results such as the surprising immunosuppressive role of anti-viral signalling pathways, and the close link between negative co-stimulation in the immunological synapse and T cell receptor trafficking. Here we review our major findings and put them into context with other published work. © 2013 Elsevier B.V. All rights reserved.

Contents 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Lentiviral vectors to genetically modify DCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Targeted activation of intracellular signalling pathways in DCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Selective targeting of intracellular signalling pathways in DCs effectively modulates T cell responses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Selective p38 activation in DCs increases anti-tumour T cell responses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Selective ERK activation in DCs for the treatment of autoimmune disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Inhibitory co-stimulation controls TCR trafficking at the immunological synapse . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Targeting intracellular signalling pathways in DCs potentiates PD-L1 silencing for cancer immunotherapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Use of lentivectors to target signalling pathways in humans: practical and ethical issues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions and future directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abbreviations: DC, dendritic cell; TAA, tumour associated antigen; NC, nucleocapsid; MA, matrix; CA, capsid; RRE, rev response element; cppt, central DNA flap; WPRE, woodchuck post-transcriptional response element; TCR, T cell receptor; pMHC, peptide–MHC complex; Th, T helper; TLR, toll-like receptor; MAPK, mitogen activated protein kinase; JNK, c-jun kinase; NF-␬B, nuclear factor kappa-light-chain-enhancer of activated B cells; ERK, extracellular signal-regulated kinase; IRF, interferon regulatory factor; OVA, ovalbumin; X-SCID, X-linked severe combined immunodeficiency; MLV, mouse leukaemia virus; Treg, regulatory T cell; MDSC, myeloid-derived suppressor cell. ∗ Corresponding author at: Navarrabiomed-Fundacion Miguel Servet, C/Irunlarrea 3, 31008 Pamplona, Navarra, Spain. E-mail addresses: [email protected], [email protected] (D. Escors). 0168-1702/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.virusres.2013.05.007

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1. Introduction

2. Lentiviral vectors to genetically modify DCs

The immune system protects our organism from a variety of threats, including pathogens, toxins and cancer. This protection is achieved through the concerted and regulated action of a number of immune and non-immune cell types. Some of these specialise in antigen capture and processing. These cells decide which of these encountered substances pose a real threat. Other cell types are specialised in neutralising pathogens and toxins, while others exert direct cytotoxicity towards infected as well as cancer cells. Above all, there are systemic homeostatic mechanisms that keep a tight control to prevent collateral damage. For example immune responses directed towards commensal bacteria living in our gut and mucosal areas would be highly detrimental. Thus, understanding the mechanisms of immune modulation in physiological and pathological conditions is essential for the development of novel, effective immunotherapies. One of the most challenging tasks of the immune system is the protection against cancer. This is a difficult task as in most cases tumour-associated antigens (TAAs) are aberrantly-expressed self-antigens, or mutated versions (quasi-antigens) (Boon and van der Bruggen, 1996; Breckpot and Escors, 2009; Campos-Perez et al., 2013; DuPage et al., 2012; Van den Eynde and van der Bruggen, 1997). Even so, there is accumulating evidence demonstrating the important role of the immune system in anti-cancer activities (DuPage et al., 2012). However, in many instances, immunotherapeutic interventions are necessary to boost these natural anti-tumour activities. Two major barriers have to be overcome to achieve efficient immunotherapy (Breckpot and Escors, 2009). The first barrier consists of breaking the natural immunological tolerance to TAAs. The second major barrier is tumour-induced immune suppression (Breckpot et al., 2003). Tumours are capable of inducing a generalised systemic immunosuppression by a variety of mechanisms, which explains the failure of many immunotherapy treatments. In fact, patients usually undergo immunotherapy in advanced stages of cancer, associated with a strong systemic immune suppression. The opposite situation occurs in autoimmune disorders. Tolerance towards self-antigens is already broken, and the major challenge is restoring the physiological immune tolerance. This is already a hard task, comparable to raising anti-tumour responses. In many instances the autoantigen is unknown, such as in rheumatic disorders (Flores-Borja et al., 2008). In recent years, palliative therapy for these diseases has improved survival and quality of life, particularly by the application of biological agents which neutralise pro-inflammatory cytokines (Bongartz et al., 2006; Nadkarni et al., 2007). Dendritic cells (DCs) have been our therapeutic target as they are major regulators of immune responses (Breckpot and Escors, 2009; Escors et al., 2008; Goold et al., 2011) and they control T and B cell responses by stimulating or inhibiting them (Tarbell et al., 2006). DCs are a heterogeneous myeloid lineage of professional antigen presenting cells with high phagocytic capacities and antigen processing/presentation capabilities (Breckpot and Escors, 2009). They patrol peripheral tissues sampling the environment, and after encountering pathogens at sites of inflammation, they take up antigen and undergo a complex phenotype/functional change (maturation). The maturing DCs migrate to secondary lymphoid organs where they present antigens to lymphocytes. Depending on the nature of the particular antigen, as well as the context in which it was found, they will trigger different types of responses (Hawiger et al., 2001). The detailed characterisation of the molecular mechanisms of DC function will help develop methods to effectively control immune responses (Steinman and Banchereau, 2007).

Lentiviral vectors (lentivectors) are excellent tools for biomedical research, as they can transduce dividing and quiescent cells (Escors and Breckpot, 2010; Escors et al., 2012; Goyvaerts et al., 2013). Importantly, lentivectors can accommodate in their lipid “viral” envelopes a wide range of glycoproteins, so they are relatively easy to pseudotype and in this way, control their in vivo tropism (Escors and Breckpot, 2010; Goyvaerts et al., 2013). Furthermore, they can stably integrate in the cell genome, leading to prolonged long-term transgene expression. In some cases, integration-deficient lentivectors can also be engineered, when integration is not required, such as in immunisation protocols (Karwacz et al., 2009). This greatly reduces the chances of genotoxicity (Escors and Breckpot, 2010). Finally, lentivectors have been successfully used in clinical trials for the correction of adrenoleukodystrophy, ␤-thalassaemia, and leukaemia (Cartier et al., 2009; Cavazzana-Calvo et al., 2010; Kalos et al., 2011; Porter et al., 2011). The first clinical trial with therapeutic lentivectors was for the treatment of HIV-1, and it has shown no adverse secondary effects so far (Levine et al., 2006; McGarrity et al., 2013; Waehler et al., 2007). Nevertheless, lentivectors integrate their genome close to transcriptionally active sites, and may cause insertional mutagenesis and gene expression through aberrant splicing (Cesana et al., 2012; Ginn et al., 2010; Knight et al., 2010). Moreover, insertional mutagenesis and genetic instability have been serious genotoxic complications of gene therapy using ␥retrovirus vectors (Hacein-Bey-Abina et al., 2003; Howe et al., 2008; Ott et al., 2006; Stein et al., 2010; Thrasher et al., 2006). Thus, their application in human therapy must be performed with caution. Lentivectors are usually engineered from the HIV-1 genome. The HIV-1 genome is made of a diploid single-stranded RNA genome that is reverse-transcribed to a single DNA molecule and stably integrates into the cell genome. This integrated version is called a provirus, and consists of a set of genes flanked by two longterminal repeats which are divided in three functional regions (Fig. 1A); The U3 region is the HIV promoter, followed by the R and U5 regions, required for reverse transcription and efficient gene expression. A key functional element is the packaging signal ( ), which is necessary for the specific packaging of the genomic RNA transcript during the assembly of the viral particle. Then, going from the 5 to the 3 end, we find the Gag-Pol gene, encoding a polyprotein made up of the nucleocapsid (NC), matrix (MA), capsid (CA)-encoding domains and following a translational frameshift, the protease, reverse transcriptase and integrase enzymes (Jacks et al., 1988; Katz and Skalka, 1994). All these genes are absolutely required for lentivector replication and assembly. The Env gene encodes the HIV envelope glycoprotein, made of two regions, the transmembrane and globular domains. The HIV genome contains other regulatory genes involved in virulence, RNA transcription, processing and transport. From these, the most important ones for engineering lentivectors are rev and tat, required for regulation of splicing and gene transcription (Feng and Holland, 1988; Katz and Skalka, 1994; Rimsky et al., 1988). The presence of these regulatory genes differentiates complex retroviruses (lentiviruses such as HIV-1, HIV-2, spumaviruses) from their simple counterparts such as ␥-retroviruses. The engineering of lentiviral vectors is straightforward. There are several published reviews describing lentivectors and their different generations (Breckpot et al., 2003; Escors and Breckpot, 2010; Zufferey, 2002). Briefly, to construct a lentivector, the majority of the HIV genes are removed leaving the LTRs and packaging signal (Fig. 1B). Other regulatory elements are included to enhance their production, such as the rev response element (RRE) (Daugherty et al., 2010), central DNA flap (cppt) (Sirven

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Fig. 1. HIV genome and lentivector structure. (A) The HIV genome is represented in this scheme. It contains the Gag-Pro-Pol and Env genes, including the regulatory/virulence accessory genes Vif, Vrp, Vpu, Nef, rev and Tat. The genome is represented as the integrated provirus, flanked by the two long-terminal repeats (LTR). On the left LTR, the subdivision into the U3, R and U5 regions is shown. , packaging signal; RRE, rev response element (B) A prototypical lentivector structure is depicted. It presents the two flanking LTRs, an internal promoter and gene of interest in addition to other elements that increase lentivector titre, such as RRE, the central polypurine flap (cPPT) and the woodchuck post-transcriptional response element (WPRE).

et al., 2000) and the hepadnavirus woodchuck post-transcriptional response element (WPRE) (Zufferey et al., 1999). Then, a promoter and transgene of interest are inserted in the lentivector construct. A large combination of promoters and genes has been used in experimental models and gene therapy. This allows the transcriptional control of gene expression in different cell types and tissues, as well as the use of regulatable expression (Escors and Breckpot, 2010). Lentivector particles are usually produced by cotransfection of the transfer (lentivector) plasmid with a number of plasmids expressing in trans the structural/regulatory proteins and an envelope glycoprotein (Fig. 2A). The plasmid encoding a (usually) viral glycoprotein allows the pseudotyping of the lentivector particle. A wide range of envelope glycoproteins from many

different virus species have been used so far, and reviewed elsewhere (Escors and Breckpot, 2010). Pseudotyping lentivectors with different envelopes allows their specific targeting to many cell types, usually coinciding with the natural tropism of the virus envelope glycoprotein. In some cases, these glycoproteins can be modified or combined with molecules that modify the original tropism. VSV-G is by far the widest used envelope, as it is a pan-tropic envelope that allows transduction of many human and animal cell types, including DCs (Akkina et al., 1996; Burns et al., 1993; Coil and Miller, 2004). Lentivector preparations can be directly used to modify cells ex vivo, or injected into the experimental animal model for either gene therapy or immunisation (Fig. 2B).

Fig. 2. Production and use of lentivectors. (A) In the scheme, the three most used lentivector production plasmids are represented in a simplified fashion; the lentivirus vector plasmid on top (transfer plasmid), and the packaging/envelope plasmids below. The packaging plasmid expresses the structural and enzymatic genes from HIV required for reverse transcription, integration and assembly. It usually incorporates Tat and Rev in second generation lentivector systems. The envelope plasmid usually encodes viral envelope glycoproteins to confer specific tropisms to the lentivector particle. The most used envelope gene is the vesicular stomatitis virus G (VSV-G) protein. (B) To generate lentivector stocks, the three plasmids are co-transfected in 293T cells (left). Lentivector particles are released in the supernatant (centre), which are collected, concentrated, and directly used for ex vivo transduction of target cells or in vivo vaccination of animal models (right).

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Fig. 3. Three signal model for antigen presentation to T cells. The three signal model of antigen presentation is depicted in this scheme. On the left, an antigenpresenting DC is shown, with MHC complexed to antigen peptides (MHC in the figure). This MHC-peptide complex is recognised by the TCR on a T cell (right), delivering “signal 1” to the T cell as shown in the figure. In addition, positive and negative co-stimulation takes place simultaneously. Positive co-stimulation is represented in this figure as the binding between CD80 on the DC surface and CD28 on the T cell surface. Negative co-stimulation is represented as the binding between PD-L1 and PD-1. The signal 1 and signal 2 (integration between positive and negative costimulatory interactions) will activate T cells. In addition, the presence of cytokines during antigen presentation (top) delivers a third signal. This “cytokine priming” will determine T cell polarisation towards different immune responses.

3. Targeted activation of intracellular signalling pathways in DCs Effective immune responses against infectious diseases and cancer rely on the differentiation between “pathogenic”/“dangerous” antigens from self-/innocuous antigens. To explain this discrimination, “the three signal” model of antigen presentation has been put forward (Iwasaki and Medzhitov, 2004; Pasare and Medzhitov, 2004) (Fig. 3). Both pathogenic and innocuous antigens are specifically recognised by T cells through binding of their T cell receptor (TCR) with a complex between major histocompatibility molecules and antigenic peptides (pMHC) on the surface of DCs. This interaction is considered “signal 1”. However, this interaction is not sufficient to activate T cells. In fact, following stimulation through signal 1 alone drives T cells into anergy, or cell death, which is necessary to keep immunological tolerance towards self-antigens (Jeon et al., 2004). To achieve full activation and proliferation, T cells need to be further stimulated by additional interactions between co-stimulatory molecules on the DC surface, and their ligands on the T cell. This interaction provides “signal 2” and takes place when “pathogenic” antigens are presented from activated (mature) DCs to T cells. Some of these interactions are activatory, such as CD80/CD86 with CD28, CD40 with CD40L, and others inhibitory, such as CD80 with CTLA-4 or PD-L1 with PD-1 (Chiang et al., 2000; Escors et al., 2012, 2008; Freeman et al., 2000; Janeway and Bottomly, 1994; Karwacz et al., 2012; Krummel and Allison, 2011; Liechtenstein et al., 2012a,b; Sansom, 2000). The integration of these antagonistic interactions will determine the level of T cell activation (Liechtenstein et al., 2012b). If activatory interactions are prevalent, then T cells will acquire effector cytotoxic activities. If inhibitory interactions are predominant T cells will differentiate into regulatory T cells, or go into anergy, exhaustion or apoptosis. This type of control maintains peripheral immunological tolerance towards innocuous/self-antigens while at the same time ensuring specific and effective T cell responses. In addition to these two signals, the type of cytokines present during antigen presentation will modify T cell differentiation into

Fig. 4. Intracellular signalling pathways controlling DC functions. DCs present on their surface a wide variety of receptors (top). Binding to their corresponding ligands will trigger a complex network of signalling molecules, which will integrate in a few well characterised pathways as shown in the figure. These pathways are largely responsible for regulating DC maturation and antigen presenting activities; NF-␬B, MAPK and IRF3 pathways. Their involvement in DC functions is indicated in the figure (bottom). The NF-␬B pathway can be specifically activated by the expression of KSHV vFLIP or NIK proteins, as depicted in the figure, while MAPK p38, JNK and ERK by their upstream MKKs. Constitutively active mutants of these upstream kinases were used to activate DCs using lentivectors.

different types. This is the “third signal” or “cytokine priming”. Examples are T helper (Th) 1, Th2, Th17 or Tregs, which are involved in cytotoxic and antibody responses, early inflammation or immunosuppression, respectively (Celli et al., 2005; Curtsinger et al., 2003a,b; Kapsenberg et al., 1999; Liechtenstein et al., 2012a; Pasare and Medzhitov, 2004). Thus, T cell responses are tightly regulated, particularly during antigen presentation, and consequently, T cell activation and differentiation can be controlled at the immunological synapse by modifying DC functions (Breckpot et al., 2007; Breckpot and Escors, 2009; Escors and Breckpot, 2010). To achieve immunological control, we have been genetically modifying DCs with lentivectors, particularly by targeting their intracellular signalling pathways (Arce et al., 2011a, 2012; Breckpot et al., 2003; Dufait et al., 2013; Dullaers et al., 2006; Goyvaerts et al., 2013; Liechtenstein et al., 2012b). DCs present on their surface a wide range of different receptors, including cytokine, growth factor, complement receptors and toll-like receptors (TLRs) (Fig. 4). Of all these, TLRs are particularly important for T cell responses, as their ligands are usually pathogen-derived molecules and trigger the up-regulation of costimulatory molecules in DCs, which provide a strong signal 2 to T cells (Breckpot et al., 2010; Hedayat et al., 2011; Hou et al., 2008; Kawai and Akira, 2008; Rakoff-Nahoum and Medzhitov, 2009). When TLR ligands bind their receptors, they trigger an intricate network of intracellular signalling pathways, which integrate and converge into just a few key signalling pathways including nuclear factor kappa-light-chain-enhancer of activated B cells (NF-␬B), mitogen activated protein kinase (MAPK) and interferon regulatory factor (IRF) pathways. The MAPK pathways are further subdivided mainly in three types, namely p38, c-jun kinase (JNK) and extracellular signal-regulated kinase (ERK) (Fig. 4). While NF-␬B, p38 and JNK are linked to inflammation and secretion of pro-inflammatory cytokines, the ERK pathway is usually involved in survival and tolerance. IRFs are transcription factors that are phosphorylated in the cytoplasm, dimerise, translocate to the nucleus and transactivate anti-viral genes (Nguyen et al., 1997).

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Fig. 5. Selective activation of signalling pathways in DCs differentially control T cell responses. In this figure, the summary of our results is shown. (A) The lentivector structure used for DC modification is shown in the figure. SFFV, spleen focus-forming virus promoter. Activator, genes encoding constitutive activators of signalling pathways. Ubiquitin pr, human ubiquitin promoter. The model antigens are expressed under the control of this promoter. (B) DCs (left) can be directly modified with lentivectors to selectively activate the indicated signalling pathways. This targeted activation will modify the DC maturation phenotype and secretion of immunomodulatory cytokines as depicted. Thus, different outcomes of T cell responses are obtained, leading to either immunisation or tolerance, and shown from left to right.

NF-␬B, MAPK and IRF pathways are simultaneously activated after TLR-ligand engagement (Kawai and Akira, 2008). We suspected that genetic activation of these pathways instead of TLR-driven activation could control DC functions and drive T cell responses in a desired direction. Thus, we expressed wellcharacterised constitutive activators of intracellular signalling pathways under the control of a ubiquitous promoter to selectively activate each one (Enslen et al., 1998; Escors et al., 2008; Lei et al., 2002; Pages et al., 1994; Raingeaud et al., 1996; Servant et al., 2003) (Fig. 4). In addition, we included a second promoter to co-express reporter and antigen genes (Arce et al., 2012; Escors et al., 2008) (Fig. 5A). To activate p38, a MKK6 mutant was used containing mutations in its activation loop (Enslen et al., 1998). An MMK7-JNK1 fusion construct was expressed to activate the JNK1 pathway (Tournier et al., 1997). To activate ERK, a MEK1 mutant was used which contained mutations in its activation loop and a deletion of its nuclear export signal (Escors et al., 2008; Pages et al., 1994). A similar approach was used to activate the IRF3 pathway, by using a mutant containing two activatory mutations in its carboxy-terminus (IRF3 2D) (Servant et al., 2003). These constitutive activators were expressed efficiently in lentivector-modified DCs and caused differential maturation phenotypes (Escors et al., 2008). Specific p38 activation increased the expression of important activatory co-stimulatory molecules such as CD80, CD40 and ICAM I, in agreement with other studies (Ardeshna et al., 2000; Arrighi et al., 2001; Sato et al., 1999; Yu et al., 2004) (Fig. 5B). However, even though p38 activation is extensively linked to secretion of pro-inflammatory cytokines, its activation in the absence of other signalling pathways did not result in production of IL-12, TNF-␣ or IL1␤ (Arce et al., 2012; Escors et al., 2008). This surprising result could be explained by low-level NF-␬B activation after lentivector transduction that otherwise takes place simultaneously following TLR engagement. NF-␬B translocation is necessary for transcriptional up-regulation of many cytokine genes, while p38 seems to open up the chromatin structure to allow NF-␬B to bind to promoters (Saccani et al., 2002). In agreement with this, NF␬B activation in DCs by expressing NF-␬B inducing kinase (NIK) with adenovirus vectors substantially increased secretion of proinflammatory cytokines such as IL12, TNF-␣ and other chemokines

(Andreakos et al., 2006). Similarly, expression of KSHV vFLIP in DCs, a constitutive activator of NF-␬B, resulted in DC maturation and a high level secretion of IL-12 and TNF-␣ (Karwacz et al., 2009; Rowe et al., 2009; Shimizu et al., 2011). Lastly, similar results were obtained by silencing the NF-␬B negative regulator A20 (Breckpot et al., 2009), which in combination with TLR stimulation enhanced cytokine production. Interestingly, it also increased IL10 expression levels, an immunosuppressive cytokine (Breckpot et al., 2009). Ex vivo JNK1 activation in DCs showed a similar result to the p38 activator albeit at lower levels (Escors et al., 2008). On the other hand, specific ERK activation strongly down-modulated CD40, while no apparent effects over the DC maturation phenotype were observed by selective IRF3 activation (Escors et al., 2008) (Fig. 5B). This was in agreement with the observation that the ERK pathway is anti-inflammatory (Agrawal et al., 2006; Arce et al., 2011b, 2012; Dufait et al., 2012; Qian et al., 2006). Interestingly, even though IRF3 expression in DCs moderately increased IFN-␤ secretion as expected, it also strongly up-regulated IL10 secretion (Escors et al., 2008) (Fig. 5B). In fact, IFN-␤ and IL10 production were found to be regulated by TLR stimulation following a common pathway that depends on TRIF-TRAF3 (Hacker et al., 2006). Moreover, we obtained evidence suggesting that IL10 expression was caused after direct binding between activated IRF3 with MYD88. This would explain at the molecular level the link between TLR-stimulated TRAF3 leading to IRF3 activation, which would then associate to MYD88, ending up with MYD88-IRF3-dependent IL10 and IFN-␤ production (Chang et al., 2007; Escors et al., 2008; Hacker et al., 2006). Thus, IL10 would then be regulated by a MYD88-dependent mechanism, and indirectly by TRIF. Surprisingly, selective ERK activation in DCs did not increase IL10 production. This is surprising as there is extensive experimental evidence demonstrating the opposite when using MEK inhibitors after TLR stimulation (Dillon et al., 2006; Qian et al., 2006; Saraiva and O’Garra, 2010; Slack et al., 2007). In fact, ERK activation did result in significant bioactive TGF-␤ expression, a pleiotropic cytokine required for the differentiation of inducible Foxp3 CD4 regulatory T cells (Tregs) (Arce et al., 2011b, 2012; Escors et al., 2008; Luo et al., 2007; Peng et al., 2004).

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Fig. 6. Immunomodulation by vaccination with lentivectors encoding activators of intracellular signalling pathways. (A) The lentivector structure used for DC modification is shown in the figure. SFFV, spleen focus-forming virus promoter. Activator, genes encoding constitutive activators of signalling pathways. Ubiquitin pr, human ubiquitin promomter. IiOVA, fusion gene between the MHC II invariant chain (Ii) and chicken ovalbumin (OVA). (B) On top, vaccination regime. Mice were vaccinated subcutaneously with lentivectors and from day 7 onwards, OVA-specific CD8 T cell responses were evaluated by IFN-␥ ELISPOT, as shown below. Note the increase in number and intensity of IFN-␥ spots when the p38 activator was co-expressed with OVA. In contrast, a decrease in spots which reflect an impaired expansion of OVA-specific CD8 T cells when the ERK and IRF3 activators were used.

4. Selective targeting of intracellular signalling pathways in DCs effectively modulates T cell responses Specific, single activation of MAPK/IRF3 intracellular pathways resulted in different DC phenotypes and in the case of ERK/IRF3, secretion of strong immunosuppressive cytokines (Escors et al., 2008). Therefore, we tested the activation of each pathway with simultaneous expression of an immunogenic model transgene in a vaccination model (Fig. 6A). For this, we used fusion construct between the amino-terminus of the MHC II invariant chain (Ii) and chicken ovalbumin (OVA) (Arce et al., 2011b; Escors et al., 2008; Rowe et al., 2006) (Fig. 6A). The IiOVA construct contains well defined class I and class II epitopes, which allowed the monitoring of CD8 and CD4 T cell responses. Additionally, the Ii chain directs the transgene to the MHC class II processing machinery, enhancing CD4 responses (Rowe et al., 2006). Interestingly, while subcutaneous vaccination with a lentivector expressing either the p38 or the JNK1 activators significantly increased OVA-specific CD8 and CD4 T cell expansion, ERK and IRF3 activation strongly suppressed them (Escors et al., 2008) (Fig. 6B). This was surprising considering the lack of secretion of pro-inflammatory cytokines after p38 or JNK1 selective activation. This suggested that either cytokines such as IL12 or TNF-␣ were not required for antigen presentation, or that these cytokines may still be produced at high levels in vivo. The latter hypothesis is likely as increased co-stimulatory molecules in DC will enhance DC-T cell association and cause “reverse signalling” in DCs leading to pro-inflammatory cytokine expression (Arce et al., 2012). In addition, the lentivector particles possess adjuvant activities that may increase immunogenicity (Breckpot et al., 2010). These results were not restricted to OVA epitopes. Vaccination of HLA-A2 transgenic mice with lentivectors co-expressing signalling activators with the human tumour antigen NY-ESO-1 showed the same results (Escors et al., 2008). MAPK p38 mediated enhancement of NY-ESO-1-specific CD8 T cell responses, and

nearly complete suppression of CD8 T cell expansion was caused by constitutive ERK or IRF3 activation (Fig. 7). The strong inhibition of T cell expansion observed after ERK activation may have been caused by CD40 down-modulation coupled with TGF-␤ production. In fact, systemic expansion of Foxp3 CD4 T cells took place after restimulation of splenocytes with class II OVA peptides (Arce et al., 2011b; Escors et al., 2008). On the other hand, the strong T cell suppression after IRF3 activation was unexpected (Figs. 6 and 7). This pathway is essential to trigger anti-viral immunity (Lin et al., 1998; Nguyen et al., 1997; Schafer et al., 1998). It is likely that the associated IL10 production may have counteracted any immunostimulatory effect. IFN-␤ transcription requires simultaneous binding of NF-␬B, AP-1 and phosphorylated IRF3 in its promoter (Thanos and Maniatis, 1995). Thus, the lack of NF-␬B and AP-1 may have dampened down the adjuvant effects of IRF3 activation. Interestingly, type I IFNs are given as a first line therapeutic agent against multiple sclerosis, as these IFNs are immunosuppressive in certain situations (Billiau, 2006; Comabella et al., 2002). 5. Selective p38 activation in DCs increases anti-tumour T cell responses We demonstrated that DC function could be modulated by lentivector targeting of intracellular signalling pathways (Escors et al., 2008). In some cases, adjuvant activities were evident after vaccination of naïve mice with model antigens (Figs. 6 and 7). However, the achievement of anti-tumour immunity is an experimental and clinical challenge, particularly in a therapeutic context. Thus, we tested p38-activated DCs expressing OVA as a cellular vaccine against an aggressive class II-negative lymphoma model, EG7. This model derives from the thymoma EL4 cell line, and stably expresses chicken OVA as a xenoantigen (Rowe et al., 2006). Many studies on tumour vaccination rely on protection studies. In protection studies, specific T cell responses are induced first

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Fig. 7. Immunomodulation of T cell responses directed towards a human TAA by vaccination with lentivectors encoding activators of intracellular signalling pathways. (A) Same as in Fig. 6A, but including the human TAA NY-ESO-1 instead of IiOVA. (B) Same as in Fig. 6B. Note the strong inhibition of CD8 T cell responses when the ERK and IRF3 activators were used, in comparison to p38 activation.

before the experimental model is challenged with cancer cells. This approach is perfectly valid if endogenous TAAs are used. However, it is not entirely relevant in cancer models expressing immunogenic xenoantigen such as OVA. In this last case, a therapeutic approach is preferred, in which cancer cells are first injected and allowed to grow before vaccination. Three major barriers have to be overcome in a therapeutic setting; (i) actively growing tumours are strongly tolerogenic, (ii) T cell responses take at least one week to reach a peak of activity; and (iii) growing tumours have increasing chances of escaping from the immunological attack (immune escape). Taking into consideration these points, we tested our p38 activator in a therapeutic experimental setting because it showed the highest adjuvant activities. As expected, tumour growth started soon after cell transfer, and regression started occurring 7 days after vaccination (Escors et al., 2008). Increased survival was evident, and in some cases, tumours completely regressed. However, even after complete regression the tumours re-appeared as some EG7 cells had lost OVA expression and thereby provided no target for the immune response. Thus, even though p38 activation increased anti-tumour T cell responses, these responses were unable to prevent immune escape (Arce et al., 2012). These results showed that p38 alone was not sufficient for tumour erradication, and that other strategies have to be applied to improve our immunisation strategies (Arce et al., 2012; Dufait et al., 2013; Liechtenstein et al., 2012b). Nevertheless, p38 activation was also effective in raising NY-ESO-1-specific T cell responses in a transgenic HLA-A2 mouse model (Fig. 7). This mouse model is frequently used in preclinical evaluation of human immunotherapy (Firat et al., 1999), but it has to be mentioned that in this mouse model, there is no immunological tolerance towards human antigens such as NYESO-1. Importantly, p38 activation in human monocyte-derived DCs (MoDCs) effectively expanded autologous MELAN-A-specific human CD8 T cells ex vivo (Escors et al., 2008). This result strongly suggested that DC-specific p38 activation could be applied as a preventive vaccination strategy for melanoma. 6. Selective ERK activation in DCs for the treatment of autoimmune disorders While specific NF-␬B, p38 and JNK1 activation enhanced immune responses, selective ERK and IRF3 activation suppressed T cell responses (Figs. 6 and 7). Therefore, we tested whether specific ERK activation could be applied for the treatment of an autoimmune disorder, and characterise the mechanisms triggered by this signalling pathway in DCs (Arce et al., 2011b, 2012). ERK is a major regulatory pathway that controls many cellular processes, such

as proliferation, growth, survival, and immune responses (Boulton and Cobb, 1991; Boulton et al., 1991; Sato et al., 1999). According to published data and our own studies, ERK activation is involved in IL10 and TGF-␤ production, two potent immunosuppressive cytokines (Arce et al., 2011b; Dufait et al., 2012; Escors et al., 2012). Interestingly, ERK activation is strongly linked to cancer and tumour-induced immune suppression (Breckpot and Escors, 2009; Nishioka et al., 2008; Roberts and Der, 2007). Therefore, we could consider tumours a mechanistical model for the induction of immunological tolerance. In particular, ERK is activated in tumour-associated M2 macrophages, tolerogenic DCs, and probably in myeloid-derived suppressor cells (MDSCs) (Dufait et al., 2012; Jackson et al., 2008). Efficient cancer immunotherapy has to counteract the strongly suppressive functions of these cell lineages. We had already observed a strong inhibition of antigen-specific T cell responses after direct subcutaneous administration of a lentivector co-expressing the ERK activator with IiOVA and NYESO-1 antigens (Figs. 6 and 7). An increase in systemic Foxp3 Tregs was demonstrated following restimulation of splenocytes from these immunised animals (Escors et al., 2008). In contrast, when a MEK1 dominant negative mutant was used, CD8 T cell expansion was boosted compared to a vaccination control. This result is consistent with the immunosuppressive activities of ERK in DCs (Arce et al., 2011b; Escors et al., 2008). Surprisingly, direct administration of the ERK activator completely prevented antigen-specific CD4 T cell expansion, leading to Treg differentiation (Arce et al., 2011b). More importantly, these differentiated Tregs were strongly boosted after a second antigen encounter in inflammatory conditions. The expanded Tregs exerted their inhibitory activities towards effector CD8 T cells in an antigen-specific manner by suppressing their expansion as well as IFN-␥ production (Arce et al., 2011b). In fact, transducing BM-DCs ex vivo with lentivectors expressing the ERK activator and their subsequent adoptive transfer in naïve mice mediated in vivo immune suppression and Treg expansion. This immune suppression was in fact mediated by the production of bioactive TGF-␤ (Fig. 5), and silencing of this cytokine using a microRNA reverted the tolerogenic activities of the ERK activator (Arce et al., 2011b). Moreover, TGF-␤ silencing had strong T cell stimulatory activities. These results were in agreement with other published experimental systems that are not restricted to DCs, in which TGF-␤ silencing has immunostimulatory properties (Conroy et al., 2012; Wei et al., 2012). Surprisingly, IL10 expression by DCs was not required for ERK tolerogenic activities. DCs from IL10 KO mice (Kuhn et al., 1993) expressing the ERK activator strongly inhibited T cell responses and enhanced Treg expansion (Arce et al., 2011b). Nevertheless, IL10

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is a potent tolerogenic/suppressive cytokine, but not necessarily the IL10 expressed by DCs. Indeed, IL10 expression by Tregs rather than DCs controlled experimental asthma (Henry et al., 2008). Our results would agree with this data and highlight that in the context of ERK activation, DC-derived IL10 seems to be unnecessary. Human ERK-activated monocyte-derived DCs behaved in a similar manner to mouse BM-DCs as Tregs are induced through secretion of bioactive TGF-␤. In addition, direct subcutaneous administration of the ERK activator suppressed inflammation in a BSA-dependent mouse model for inflammatory arthritis. The ERK activator tolerised mice towards OVA, and intra-articular coadministration of OVA together with BSA was sufficient to prevent knee inflammation in BSA-sensitised mice (Arce et al., 2011b). This therapeutic strategy strongly boosted Treg expansion in the local draining lymph nodes, and inhibited Th1 and Th17 inflammatory cells. Mononuclear infiltration in the articular capsule was thereby avoided, which prevented destruction of articular cartilage and bone (Arce et al., 2011b).

7. Inhibitory co-stimulation controls TCR trafficking at the immunological synapse In our studies p38 activation and also ERK inhibition in DCs enhanced T cell responses. Importantly, p38 activation in DCs could control tumour growth to some extent and increase survival. Unfortunately, this strategy did not establish effective tumour eradication in our lymphoma experimental model. We then tested whether enhancing co-stimulation during antigen presentation could improve anti-tumour immunity, using lentiviral vectors to modify DCs. In recent years, blockade of the interaction between PD-L1 on cancer cells with PD-1 on T cells has been attracting the interest of researchers and clinicians (Karwacz et al., 2012; Liechtenstein et al., 2012a,b; Sakthivel et al., 2012). Blocking antibodies are routinely used in experimental models, and they have already been tested in human clinical trials for the treatment of a number of cancers (Brahmer et al., 2012; Topalian et al., 2012). Blocking this interaction hyperactivates T cells and induces high level secretion of pro-inflammatory cytokines (Hobo et al., 2010; Karwacz et al., 2011; Liechtenstein et al., 2012a). As PD-L1 is nearly ubiquitously expressed in all tissues, this interaction is critical to maintain peripheral tolerance. PD-1 expression that is upregulated in activated T cells may prevent autoimmune attack against selftissues (Fife et al., 2009; Latchman et al., 2004). Promising results have been obtained in human clinical trials, although associated to serious side effects directly caused by inflammatory disorders (Brahmer et al., 2012; Topalian et al., 2012). Silencing PD-L1 expression in antigen-presenting DCs in a targeted and specific manner, as well as testing its effects over antigen presentation to T cells was the next step. Simultaneous delivery of IiOVA and a PD-L1-targeted microRNA using lentivectors reduced DC PD-L1 expression in half, and was enough to exert significant effects on T cells (Karwacz et al., 2011). We noticed that OVA-specific CD8 T cells strongly associated to PDL1-silenced OVA-presenting DCs. Strikingly, ligand-induced TCR down-modulation was inhibited in these CD8 T cells (Karwacz et al., 2011). This inhibition correlated to lack of expression of Cbl-b, an E3 ubiquitin ligase implicated in TCR down-modulation and termination of TCR signal transduction (Bachmaier et al., 2000; Chiang et al., 2000; Jeon et al., 2004; Marmor and Yarden, 2004; Naramura et al., 2002). Ligand-induced TCR down-modulation is a well-known process by which T cells remove their TCRs from the surface shortly after antigen presentation (Liu et al., 2000). We put forward the “extrinsic signal model for antigen-induced TCR down-modulation” (Escors et al., 2011; Karwacz et al., 2012, 2011; Liechtenstein et al., 2012a,b) (Fig. 8). The physiological

reasons for this phenomenon remain unclear (Escors et al., 2012). Ligand-induced TCR down-modulation might control excessive T cell activation, and/or could be required for T cells to acquire effector functions (Karwacz et al., 2012; Liechtenstein et al., 2012a). In our experimental system we confirmed in agreement with other published work that the resulting TCRhigh CD8 T cells were hyperactived and exhibited increased proliferation and production of IFN-␥ and IL17 (Hobo et al., 2010; Karwacz et al., 2011; PilonThomas et al., 2010). Similar T cell phenotypes have been observed in Cbl-b KO mice (Hinterleitner et al., 2012; Loeser et al., 2007; Naramura et al., 2002; Paolino et al., 2011; Shamim et al., 2007). These hyperactivated T cells caused enhanced destructive inflammation in an OVA-dependent inflammatory arthritis mouse model while exhibiting faster anti-tumour attack (Karwacz et al., 2011). Tumours from treated mice regressed earlier and were smaller than tumours in control immunised mice. However, to our surprise earlier regression did not result in improved survival. Cure rates were the same as control mice with non-silenced PD-L1 (Karwacz et al., 2011). This surprising result suggested that TCRhigh T cells were attacking the tumour early in the T cell expansion phase, but possibly not reaching the “critical mass” necessary for a “blitz” anti-tumour attack (Bricogne et al., 2012). This premature cytotoxic activity was therefore insufficient to achieve tumour eradication. We proposed the “critical mass” hypothesis of ligand-induced TCR down-modulation to provide a physiological interpretation of this phenomenon (Bricogne et al., 2012) (Fig. 9). TCR down-modulation takes place during the first 10 days after antigen presentation, reaching a maximum on days 4–5. TCR surface expression gradually recovers after day 5 until the original surface expression levels are reached, when T cell expansion has reached its maximum. At this stage T cells possibly recover their full effector activity. T cells can then migrate to inflamed/infected sites and exert their full cytotoxic attack leading to efficient eradication of infected/malignant cells (Bricogne et al., 2012).

8. Targeting intracellular signalling pathways in DCs potentiates PD-L1 silencing for cancer immunotherapy As already discussed above, PD-L1 silencing hyperactivates effector CD8 T cells and leads to a fast anti-tumour immune response. This early anti-tumour attack can be of relevance for tumour immunotherapy, in its race against tumour growth and metastasis (Bricogne et al., 2012). However, similar to p38 activation, it was not sufficient for tumour eradication. In other experimental systems, PD-L1/PD-1 blocking has been effective in combination with other immunostimulatory strategies or low-dose chemotherapy (Curran et al., 2010; Dai et al., 2012; Mangsbo et al., 2010; Pilon-Thomas et al., 2010; Sierro et al., 2011). In agreement with our results, PD-L1/PD-1 blockade enhanced the association between T cells and antigen presenting cells and increased effector T cell tumour infiltration (Fife et al., 2009; Karwacz et al., 2011). Blocking antibodies have been tested in human clinical trials with some promising success, but their efficacy varies from cancer to cancer and these treatments are highly toxic. As expected, this toxicity is directly related to uncontrolled inflammatory reactions (Brahmer et al., 2012; Liechtenstein et al., 2012a; Topalian et al., 2012). To overcome all these issues we combined PD-L1 silencing with expression of the p38 constitutive activator and the ERK inhibitor. The hypothesis was that the use of these DC molecular activators would ensure the efficient T cell activation during antigen presentation, while synergising with PD-L1 silencing (Liechtenstein et al., 2012b). Interestingly, the combination of these DC activators with PD-L1 silencing further increased IFN-␥ T cell expansion, which resulted in a further decrease in tumour size, and more importantly,

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Fig. 8. Extrinsic signal model for ligand-induced TCR down-modulation. (A) DCs (top) present antigen in the context of MHC molecules (pMHC) to specific TCRs on the surface of T cells (bottom). This binding triggers TCR-dependent signalling leading to T cell activation and surface expression of PD-1. (B) PD-1 associates then with PD-L1 on the DC surface. This interaction leads to, amongst other things, transcriptional up-regulation of the E3 ubiquitin ligase Cbl-b, as shown in the figure. (C) The concerted activity of Cbl-b with SHIP phosphatases (not shown in the figure) results in termination of TCR signalling, and TCR removal from the T cell surface, as shown in the figure.

in a significant increase in survival. Cure rates (tumour-free for at least three months) of about 80% were routinely obtained in the EG7 lymphoma model (Escors et al., 2011; Karwacz et al., 2012, 2011). 9. Use of lentivectors to target signalling pathways in humans: practical and ethical issues Gene therapy in humans was attempted as early as the 1970s, where Shope papillomavirus was used to correct hyperargininaemia with papillomarivirus arginase (Escors and Breckpot, 2010). Although this clinical trial was unsuccessful, it irreversibly opened the door for the therapeutic application of gene therapy. From a therapeutic point of view, the first successful gene therapy trials were carried out in the early 2000s for the correction of Xlinked severe combined immunodeficiency (X-SCID) in children (Cavazzana-Calvo et al., 2000; Gaspar et al., 2004). In these trials, ␥retrovirus vectors derived from mouse leukaemia virus (MLV) were utilised as gene carriers to haematopoietic stem cells. The results from these trials were truly remarkable and were quickly followed by similar studies, including the correction of X-linked granulomatous disease in young adults (Ott et al., 2006). However, the success of these trials was obscured by the appearance of leukaemia in some treated children from the X-SCID trial (Hacein-Bey-Abina et al., 2003) as well as genetic instability and loss of transgene expression in the granulomatous trial (Stein et al., 2010). The adverse genotoxic effects in the X-SCID clinical trial were mainly caused by insertional mutagenesis, a phenomenon linked with retroviral vector integration close to proto-oncogenes (Hacein-Bey-Abina et al., 2003; Howe et al., 2008; Lund et al., 2002; Mikkers et al., 2002; Ott et al., 2006; Stein et al., 2010; Stocking et al., 1988; Suzuki et al., 2002). Meanwhile, new gene therapy vectors engineered mainly from the HIV-1

genome were being quickly developed. These HIV-based vectors, called lentivectors were superior to MLV-based vectors due to their capacity to transduce dividing and non-dividing cells (Dull et al., 1998; Naldini et al., 1996a,b; Trono, 2000; Zufferey et al., 1999, 1998, 1997). After the important genotoxicity exhibited by MLVbased vectors, therapeutics turned direction and increased the use of lentivectors in gene therapy. Lentivectors have been shown to be less genotoxic than their simple retrovirus counterparts, as their integration patterns are different from those of MLV vectors (Biffi et al., 2011; Bokhoven et al., 2009; Zhou et al., 2010). However, major issues of concern remain even for lentivectors, as their integration significantly alters gene expression patterns and splicing, leading to transactivation of putative proto-oncogenes (Cesana et al., 2012; Knight et al., 2010). In addition, other undefined “cellular factors” can also increase the susceptibility for retroviral/lentiviral vector toxicity (Ginn et al., 2010; Kustikova et al., 2009). Nevertheless, the first gene therapy clinical trials using lentivectors have already been successfully carried out for the treatment of HIV-1 (Levine et al., 2006), X-linked adrenoleukodystrophy (Cartier et al., 2009), ␤-thalassaemia (Cavazzana-Calvo et al., 2010) and chronic lymphoid leukaemia (Kalos et al., 2011; Porter et al., 2011). So far, no serious adverse effects have been observed in the HIV-1 trial, which was the first clinical trial utilising lentivectors (McGarrity et al., 2013). Possibly one of the major barriers to overcome for the routine application of lentivectors in gene therapy is the lack of stable producer cell lines for clinical application (Escors and Breckpot, 2010; Escors et al., 2012). Although several efficient experimental systems have been engineered over the years, so far they have not been successfully translated into a clinically acceptable product (Farson et al., 2001; Ikeda et al., 2003; Ni et al., 2005; Throm et al., 2009). GMP grade lentivector production is therefore not cost-effective

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potential is by far lower than that of largely undifferentiated haematopoietic stem cells. Moreover, the possibility of engineering integration-deficient lentivectors would speed up quick translation into human patients (Karwacz et al., 2007, 2009; Yanez-Munoz et al., 2006). The expression of constitutive activators of signalling pathways may pose an additional problem. This could be particularly the case for constitutive activators of immunosuppressive pathways for the treatment of autoimmune disease, such as ERK activators. Although active MEK1 mutants do not seem to be oncogenic on their own (Anastasaki et al., 2009), constitutive Raf/ERK activation has been observed in tumours conferring T cell suppressive activities, survival and cancer cell proliferation (Jackson et al., 2008; Nishioka et al., 2008; Pages et al., 1994; Roberts and Der, 2007; Strobeck et al., 1999). Thus, careful assessment of lentivectors expressing ERK activators is necessary before their translation into human therapy. A careful balance between the benefits versus the potential drawbacks will have to be seriously assessed. Nevertheless, the valuable data from the experimental application of ERK activators may uncover a number of interesting pharmaceutical targets. In recent years, progress has been made in the development of lentivectors that integrate in targeted, specific genomic regions, or lentivectors that can directly correct gene mutations (Gijsbers et al., 2010; Izmiryan et al., 2011; Lombardo et al., 2007; Schenkwein et al., 2013). The development of these technologies may in the near future circumvent the genotoxic potential of retroviral and lentiviral vectors.

10. Conclusions and future directions

Fig. 9. Critical mass hypothesis of T cell responses. According to our data, we have proposed this hypothesis to provide a physiological explanation to ligand-induced TCR down-modulation. The top graph represents an idealised clonal expansion of antigen-specific T cells following antigen presentation. The graph in the middle represents a model of TCR surface expression, following antigen presentation by PD-L1-silenced DCs (red) in comparison to standard DCs. Please note that the exponential T cell expansion of the top graph coincides with TCR down-modulation. When T cell expansion reaches its peak, T cells recover TCR surface expression. The graph in the bottom represents tumour growth after cellular vaccination with antigen-presenting DCs with silenced PD-L1 (red) or standard DCs. Please note that tumour regression takes place after T cell numbers have reached a maximum (blue). Interestingly, this is not the case when PD-L1 is silenced in DCs. Tumour regression starts earlier. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

yet, and it largely relies on transient transfection of producer cells (Merten et al., 2011). If the absence of important genotoxic events is confirmed from the current lentivector gene therapy clinical trials there should be less concerns for their application in vaccination. This would lead to their “routine” use in cancer immunotherapy and treatment of infectious diseases. After subcutaneous vaccination, post-mitotic and highly differentiated cells are transduced. Their oncogenic

Genetic engineering of potent antigen presenting cells, such as DCs, allows the fine-tuning of immune responses. This is particularly important in cancer immunotherapy and in the treatment of autoimmune disease. We have been researching the role of intracellular signalling pathways in DCs that modulate immune responses. However, especially for cancer immunotherapy, two major barriers have to be overcome to achieve therapeutically effective anti-tumour immune responses. The first one is the natural tolerance towards TAAs. TAAs are often auto-antigens that are overexpressed, or aberrantly expressed in tumour cells. In other cases they are mutated versions of self-antigens. In any case, there are strong natural tolerogenic mechanisms in place that prevent effective anti-TAA T cell responses. The first major mechanism is clonal deletion in the thymus, called negative selection. During T cell development, the strongly auto-reactive T cells undergo apoptosis, and only auto-reactive T cells with low affinity TCRs may escape negative selection. These low affinity T cells may exert potential anti-cancer activities, if adequately activated. However, their activation is a therapeutic challenge due to their low affinity TCRs. Current research is therefore focused on the genetic modification of T cells by the introduction of TAA-specific high affinity TCRs in patient’s T cells. In this fashion therapeutic anti-tumour activities have been achieved (Morgan et al., 2006; Park et al., 2011; Parkhurst et al., 2011; Robbins et al., 2011; Thomas et al., 2007). The second major tolerogenic mechanism also occurs during T cell development in the thymus, where some high-affinity autoreactive T cells develop into natural CD4 CD25 Foxp3 regulatory T cells (nTregs) (Sakaguchi, 2003; Sakaguchi et al., 2008). The suppressive activity of this cell type is a major blockade to anti-tumour T cell responses. Deletion of these Tregs leads to spontaneous tumour regression, but also to uncontrolled inflammation and autoimmunity (Gallimore and Godkin, 2008; Hori et al., 2003; Sakaguchi et al., 2006). However, counteracting the activity of these nTregs with anti-CTLA4 antibodies (Ipilimumab in human therapy) can boost anti-tumour

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activity (Peggs and Quezada, 2010), and blocking PD-L1/PD-1 interactions is already applied in human therapy with some promising success (Liechtenstein et al., 2012a,b). While boosting natural T cell responses against cancer cells has shown promising results, it may not be sufficient in a highly systemic immunosuppressive environment as that found in advanced stages of cancer. This environment is caused by tumour-induced immune suppression and is triggered by a variety of mechanisms present in a high tumour burden environment. Myeloid differentiation from the bone marrow is altered as a consequence of high tumour burden, leading to the systemic appearance of MDSCs (Gabrilovich and Nagaraj, 2009; Peranzoni et al., 2010). These cells comprise a highly heterogeneous group usually identified in mice by the co-expression of CD11b and Gr1. These myeloid cells possess potent immunosuppressive activities which can be exerted by antigen-specific and unspecific mechanisms (Highfill et al., 2010; Li et al., 2009; Srivastava et al., 2010; Youn et al., 2013). They are roughly subdivided into two main types according to their expression of Ly6G, monocytic and granulocytic MDSCs (Peranzoni et al., 2010). However, expression of this and other markers does not define them as a suppressive myeloid lineage, as the corresponding phenotypes are also found in naïve healthy animals as inflammatory monocytes and granulocytes without suppressive activities (Youn et al., 2013). A complex variety of factors produced within the tumour environment convert these cells into MDSCs (Corzo et al., 2010; Dolcetti et al., 2010; Marigo et al., 2010; Xiang et al., 2009). Even though there are many ways in which MDSC differentiation can be achieved ex vivo, these strategies are largely inefficient, as they are cumbersome and cannot keep MDSC proliferative and differentiation potential (Lechner et al., 2010; Youn et al., 2013). It would be highly desirable to efficiently replicate MDSC differentiation ex vivo to mimic the tumour environment and maintain both MDSC proliferative potential as well as their differentiation capacities. These two characteristics are observed in vivo, and are extremely difficult to replicate ex vivo (Youn et al., 2013). It is imperative for cancer immunotherapy to successfully counteract the differentiation and activity of Tregs and MDSCs and might be even more important than finding treatments designed to boost antigen presentation and T cell effector activities (Liechtenstein et al., 2012b). Some chemotherapeutic agents such as sunitinib can achieve this by reducing MDSC and Treg expansion, while enhancing DC activities (Arce et al., 2012; Xin et al., 2009). However, the field of cancer immunotherapy would surely benefit from an MDSC differentiation system similar to the extensively used BM-DC system, or human DC differentiation from blood monocytes. These types of ex vivo systems would allow the systematic testing of current and novel immunotherapy treatments, which is necessary before their application in human therapy.

Acknowledgements TL is funded by a University College London Overseas PhD Scholarship. NPJ is funded from an APPICS scholarship from the Health Department and European Social Fund (PO Navarra 2007–2013) and the Basque Country Foundation for Health Innovation and Research (Bioef). CB is funded by a University College London MB-PhD Scholarship. AL is funded by a University College London Bench-to-Bedside PhD Scholarship. The Structural Genomics Consortium Oxford is a registered UK charity (number 1097737) that receives funds from the Canadian Institutes of Health Research, The Canadian Foundation for Innovation, Genome Canada through the Ontario Genomics Insitute, GlaxoSmithKline, Karolinska Institutet, the Knut and Alice Wallenberg Foundations, the Ontario Innovation Trust, the Ontario Ministry for Research and Innovation, Merck & Co., Inc., the Novartis Research Foundation, the Swedish Foundation

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for Strategic Research and the Wellcome Trust. Karine Breckpot is funded by the Fund for Scientific Research-Flandes. DE has been funded by an Intraeuropean Marie Curie Fellowship, an Arthritis Research UK Career Development Fellowship (18433) and currently by a Miguel Servet Fellowship from the Instituto de Salud Carlos III, Spain. References Agrawal, A., Dillon, S., Denning, T.L., Pulendran, B., 2006. ERK1−/− mice exhibit Th1 cell polarization and increased susceptibility to experimental autoimmune encephalomyelitis. Journal of Immunology 176 (10), 5788–5796. Akkina, R.K., Walton, R.M., Chen, M.L., Li, Q.X., Planelles, V., Chen, I.S., 1996. Highefficiency gene transfer into CD34+ cells with a human immunodeficiency virus type 1-based retroviral vector pseudotyped with vesicular stomatitis virus envelope glycoprotein G. Journal of Virology 70 (4), 2581–2585. Anastasaki, C., Estep, A.L., Marais, R., Rauen, K.A., Patton, E.E., 2009. 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