Human skin dendritic cells as target for anti-tumor vaccination Fehres, C.M.
2015
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Fehres, C. M. (2015). Human skin dendritic cells as target for anti-tumor vaccination.
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Understanding the biology of antigen cross-presentation for the design of vaccines against cancer Fehres CM1, Unger WWJ1, Garcia-Vallejo JJ1 and van Kooyk Y1*
1Department of Cell Biology and Immunology, VU University medical center, Amsterdam, The Netherlands
*Corresponding author Front Immunol. 2014 8;5:149
General Introduction
Chapter 1
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The skin immune system
The skin is the largest organ of the human body and forms a barrier that protects our organism from the environment. In addition to its mechanical barrier function, which restricts water and heat loss, the skin plays an important role in the protection against invading pathogens, like bacteria and viruses, and is therefore equipped with a variety of immune cells and mediators[1]. The human skin consists of two layers: the epidermis and the dermis. The epidermis is the outermost layer of the skin and consists of stratified squamous epithelium. The main cell types present in the epidermis are keratinocytes (KCs), melanocytes, which produce melanin resulting in skin pigmentation and Langerhans cells (LCs), a subset of antigen- presenting cells (APCs) that will be discussed in more detail later in this chapter. The epidermis is tightly connected to the underlying dermis by the basement membrane.
The dermis is rich in extracellular matrix and contains fibroblast, lymph and blood vessels. In addition, many different immune cells reside in the dermis, such as T cells, macrophages and distinct dermal dendritic cell subsets (dDCs)[2].
Human cutaneous DC Human skin DC subsets
Within the human skin, three main populations of DCs can be found that can be separated based on the expression of CD1a and CD14 (Figure 1). CD1ahighlangerin+ LCs reside in the epidermis and the CD14+ dDCs and CD1a+/CD1c+ dDCs are found in the dermis. All DC subsets can be found in the skin-draining lymph nodes, showing the migratory potential of these cells from the skin upon activation[3]. Epidermal LCs are derived from precursor cells residing in the skin. However, under inflammatory conditions they can also develop from monocytes recruited from the blood[4;5]. Human LCs have been shown to have varying functions. On one hand, a potent cross-priming activity and initiation of allogeneic CD8+ T cells responses in an IL-15-dependent manner is described[6;7]. Since T cell-mediated immunity is considered of importance for tumor eradication, it may be beneficial to specifically target LCs for immunotherapy. On the other hand, LCs have also been shown to contribute to the expansion of regulatory T cells, which implicate that these cells contribute to tissue homeostasis [8]. CD1a+ dDCs seem to be more efficient at antigen cross-presentation of soluble antigen; a phenomenon described later this chapter, compared to the CD14+ dDCs[6;9;10]. Dermal DCs have been shown to produce interleukin-10 (Il-10) and were able to generate Th2-mediated CD4+ T cell responses, essential for the induction of humoral immunity[11;12]. The CD4+ T cell skewing abilities of dermal DCs seem not to be fixed, but dependent on the factors present in the microenvironment, the number of dDCs and their activation state, which all determine the capacity to induce Th1, Th2 and Th17 profiles[13;14]. In addition to the three main populations of skin DCs, a minor BDCA3highCD14-CD11clow-
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int subset of DCs has been recently identified in human skin, lung and liver. Phenotypic analysis has suggested that these cells are potentially related to the blood BDCA3+ DCs. Skin BDCA3high DCs have been shown to be superior in cross-presentation of soluble antigens when compared to the other skin subsets, as well as compared to BDCA3+ DCs, BDCA1+/CD1c+ DCs and CD14+ monocytes derived from blood[15].
Care should be taken not to confuse the BDCA3high skin DCs described by Haniffa et al. with the dermal BDCA3+CD14+ DCs described by Chu et al. [16]. The latter are immunoregulatory tissue-resident DCs characterized by the constitutive secretion of IL-10. Human skin draining lymph nodes has been shown to contain migratory LCs, CD1a+ dDCs and CD14+ dDCs, indicating that these cells can migrate towards the skin-draining lymph nodes upon activation in the skin[10]. CCR-7 dependent migration to the lymph nodes has been described for LCs and CD1a+ dDCs[9;9;10;17], whereas CD14+ dDCs seem to migrate from skin without the use of lymph vessels or the expression of CCR7[9].
Expression of pattern-recognition receptors by skin DCs
DCs are equipped with a broad range of pattern-recognition receptors (PRRs) that recognize pathogen-associated molecular patterns (PAMPs) and self-derived molecules from damaged cells, also known as damage-associated molecular patterns (DAMPs). Activation of PRRs triggers downstream signaling pathways leading to the induction of innate immune responses, such as the production of inflammatory cytokines, type I interferon and other mediators[18]. Besides the activation of immediate host defense responses, activation of PRRs on DCs can also prime and orchestrate antigen-specific adaptive immune responses. Classes of PRRs expressed by DCs are Toll-like receptors (TLRs), C-type lectin receptors (CLRs), Nod- like receptors (NLRs) and RIG-I-like receptors (RLRs). Of these, the TLRs and CLRs are of main focus in this thesis and will be discussed in detail.
Toll-like receptors
The best-characterized class of PRRs are TLRs, which are able to recognize various structures of bacterial or viral origin. In humans, the TLR family is comprised of 10 members, of which TLR1, 2, 4, 5, 6 and 10 are expressed on the outer cell membrane and TLR3, 7, 8 and 9 reside within endosomal compartments[19]. Cell surface TLRs mainly recognize microbial membrane components, like for example the recognition of bacterial lipopolysaccharide (LPS) by TLR4. In contrast, intracellular TLRs recognize nucleic acids derived from bacteria and viruses. As example, TLR3 senses viral double-stranded RNA (dsRNA), small interfering RNAs and self-RNAs derived from damaged cells[18]. Upon recognition of PAMPs by the TLRs, they recruit a specific set of adaptor molecules, such as MyD88 and TRIF, and initiate downstream signaling events leading to the secretion of inflammatory cytokines, type I interferon (IFN), chemokines, and antimicrobial peptides[20]. These responses cause recruitment of neutrophils and activation of macrophages, resulting in direct killing
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Figure 1. Main dendritic cell subsets in healthy human skin. Expression levels of molecules and functions commonly used to identify and discriminate the subsets are indicated. +++ high expression; ++ moderate expression; + low expression; - negative; -/+ heterogeneous expression within the subset.
of the pathogen. Moreover, activation of TLR signaling leads to maturation of DCs, contributing to the induction of antigen-specific T cell responses.
C-type lectin receptors
CLRs are carbohydrate-recognition molecules that bind carbohydrates expressed by pathogens, commensals and endogenous proteins. Binding of a carbohydrate structure to the CLR occurs via its carbohydrate recognition domain (CRD) and may result in pattern recognition, cell-cell communication and ligand-induced
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signaling[21]. Besides, most CLRs are capable of internalizing glycosylated antigen, which is followed by routing to major histocompatibility complex (MHC) class I and II loading compartments and thereby contributing to the activation of adaptive immune responses. The CLRs discussed in this thesis, DC-specific intercellular adhesion molecule-3 grabbing non-integrin (DC-SIGN) and langerin, belong both to the type II CLRs, which hold only one CRD and an extracellular C-terminus[22]. CLRs are generally divided in two different classes: the galactose/GalNAc-binding lectins that contain a Glutamine-Proline-Aspartic acid (QPD) motif within their CRD and the mannose/fucose-binding lectins that contain a Glutamic acid-Proline-Aparagine (EPN) motif in the CRD[23]. Both DC-SIGN and langerin contain an EPN motif[24;25].
The glycan specificity, molecular orientation and function of DC-SIGN and langerin will be shortly discussed in the following sections.
DC-SIGN
DC-SIGN is probably the best characterized CLR expressed by DC. Its expression is restricted to human dermal DCs and other APCs of the myeloid lineage[26]. DC- SIGN has been shown to bind to mannosylated glycans and fucose-containing glycans, such as the Lewis type antigens, due to the EPN motif present in the CRD[27-29]. For DC-SIGN, recognition of the Lewis blood group antigens Lea, Leb, LeX and LeY is described. A schematic representation of the Lewis antigens is given in Figure 2. Binding to fucose has been described to occur with a higher affinity compared to mannose[30]. Additionally, increasing the number of glycan residues in the structure results in a higher avidity, which can compensate for lower affinity binding of DC-SIGN[30-34]. With regard to the molecular orientation, DC-SIGN has been described to form oligomers via repeats in the neck region, which allows for a high level of flexibility to the CRDs and strengthens DC-SIGN-ligand interactions[22].
In addition, the presence of DC-SIGN in nanodomains on the plasma membranes of DCs has been reported, which further optimizes the DC-SIGN-ligands interactions[35].
DC-SIGN has been shown to interact with a broad variety of pathogens, such as HIV[36], Candida albicans[37], Hepatitis C virus[38;39] and Mycobacterium tuberculosis[40]. These interactions result in the internalization of the pathogen. In this thesis, we exploited the function of DC-SIGN as antigen uptake receptor to target tumor antigens to DCs. DC-SIGN has not only been described to internalize antigens, it also facilitates the processing and intracellular routing of the antigen to MHC class I and II loading compartments, which subsequently enhances antigen- specific CD4+ and CD8+ T cell responses [41-44].
Langerin
In humans, langerin is mainly expressed by LCs. An interesting feature of langerin is its association with Birbeck granules (BG), which are rod-shaped intracellular structures induced upon langerin expression[45;46]. It has been shown that antibodies directed against langerin are internalized in BGs, providing access of
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antigen to a LC-specific non-classical antigen-processing pathway[25]. The glycan specificity of langerin shows some overlap with DC-SIGN: both receptors bind to mannosylated glycans, but langerin interacts only with the difucosylated Lewis antigens Leb and LeY[29]. Langerin has shown to recognize pathogens such as HIV, Candidia, Saccharomyces and measles virus (MV) in a glycan-dependent manner[47-49]. Targeting antigen to langerin using monoclonal antibodies (moabs) has led to the development of antigen-specific Th1 and CD8+ responses. However, langerin-mediated internalization of MV only induced MV-specific CD4+ responses, but no CD8+ T cell responses[48]. Besides, whereas DC-SIGN is organized in tetramers, langerin forms trimers through a coiled-coil structure in the extracellular neck region, leading to a rather rigid position in the membrane compared to DC-SIGN[50].
The function of skin APCs: antigen presentation and activation of T cells Induction of CD4+ and CD8+ T cell responses
DCs reside in tissues, such as the skin, where they sample the environment for incoming pathogens or changes in the local environment. After recognition of the antigen via the PRRs expressed by the DCs, the antigen is internalized, transported into intracellular compartments where the antigen is processed in peptides that are presented on MHC. These peptide-MHC complexes are subsequently transported to the cell surface of the DC, where they can be recognized by antigen-specific T cells. Simultaneously with the uptake and processing of the antigen, DC maturation occurs after PRR activation. During this maturation process, DCs will increase the expression of co-stimulatory molecules, such as CD86, CD83 and CD70, and start to produce cytokines that influence the T cell responses. Moreover, the expression of MHC molecules is upregulated, as well as the expression of the chemokine receptor CCR7, allowing migration of the DCs to the T cell areas in the draining lymph nodes.
In the lymph nodes, antigen-specific CD8+ and CD4+ T cells can recognize the presented peptides within MHC-I or MHC-II molecules, respectively, leading to their
Figure 2. Overview of the Lewis blood group antigens studied in this thesis.
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activation, proliferation and development in effector T cells[51]. Here, we briefly describe the routes of antigen loading on MHC-I and MHC-II molecules
Antigen presentation to CD4+ T cells
Internalization and processing of extracellular antigens is followed by peptide presentation in MHC-II molecules, thereby allowing the activation of CD4+ T cells via the T cell receptor. After synthesis of the MCH-II molecule in the endoplasmatic reticulum (ER), complexes are formed with CD74 (also known as the Invariant chain) to allow proper folding, trafficking and protection of the peptide-binding groove.
CD74 helps guiding the CD74-MCH-II complex move on to the endolysosomal pathway, where late endosomal proteases such as cathepsin D and L degrade CD74 and leave MCH-II complexed to the peptide-binding groove part of CD74 (CLIP protein), which is later exchanged for an antigenic peptide with the help of the chaperone HLA-DM[52]. These MHC-II-peptide complexes are transported to the DC plasma membrane and are presented to antigen-specific CD4+ T cells.
Antigen presentation to CD8+ T cells
Although the process leading to antigen presentation on MHC-I involves six basic steps; namely the acquisition of antigens (1); tagging of the antigenic peptides for destruction (2); proteolysis (3); transport of peptides to the ER (4); loading of peptides to MHC-I (5) and the display of peptide-MHC-I complexes on the cell surface (6), the variety of intracellular compartments and pathways involved in MHC-I antigen presentation is considerably more complex than that of MHC-II[53].
There are two main sources of antigens for MHC-I presentation, intracellular and extracellular. Antigenic peptides derived from intracellular, cytosolic proteins are the prime source of peptides for MHC-I, but other proteins carrying signal sequences targeting to the secretory pathway can also be presented on MHC-I[54], either from defective ribosomal products or from mature proteins[55;56]. These mechanisms are at play on all cells expressing MHC-I. However, what makes DCs and, to a lesser extent, also macrophages and B cells best at antigen cross-presentation is their capacity to use extracellular antigens as source of peptides for MHC-I presentation.
Antigen cross-presentation
The uptake of extracellular antigens by APCs is achieved by three main transport pathways, namely receptor-mediated endocytosis, phagocytosis and macropinocytosis[57;58]. Amongst the many classes of receptors that mediate endocytosis of antigens are the B cell receptor, Fc receptors, heat-shock proteins, scavenger receptors and the earlier described CLRs. In general, these receptors mediate internalization of antigens to endosomes, however, the nature of the endosomes and their fate seem to vary for the different receptor types involved, and consequently, also their efficiency in inducing antigen cross-presentation.
Cross-presentation has been described to be dependent on two molecular pathways.
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The cytosolic pathway of antigen cross-presentation is dependent on the ABC peptide transporter TAP and the proteasome[59]. The proteasome is a self- compartmentalized, energy-dependent nanomachine that works as a protease to degrade misfolded, damaged and inaccurately synthesized proteins[60]. Under inflammatory conditions, such as IFN-γ or DC maturation[61], the proteasome undergoes structural changes in its substrate-binding pockets that contribute to optimizing the quality and quantity of the generated peptides[62]. In addition, a proteasome-dependent, yet TAP-independent pathway of cross-presentation has been recently described, suggesting the existence of a still unidentified peptide transporter (Figure 3)[63].
The cross-presentation pathway referred to as vacuolar uses endolysosomal proteases to degrade internalized bacteria and other antigens, frequently particulated, in order to allow loading on MHC-I molecules, which are recycled from the extracellular membrane, in the same compartment was where the peptides were degraded[64]. Also proteasome-derived peptides may enter the vacuolar pathway[65;66]. An overview of the cytosolic and vacuolar pathway of antigen cross-presentation is depicted in Figure 3.
Which of these antigen cross-presentation pathways is followed is dependent on the nature of the antigen, the route of antigen uptake and the APC subset. For example, evidence indicates that limited antigen degradation correlates with efficient cross-presentation[67]. Primarily decreased proteolysis is found in the endocytic compartments of DCs compared the other APCs, probably due to low levels of lysosomal proteases, or decreased protease activity, contributing to the superior function of DCs with regard to cross-presentation.
Antigen cross-presentation as tool for immunotherapy
Antigen cross-presentation is an important mechanism for the development of specific CD8+ T cells directed against tumor antigens. Therefore, there has been a major focus on the use of DCs, as most potent cross-presenting cells, in cancer immunotherapy. The aim of DC vaccination is to induce tumor-specific effector T cells that can reduce the tumor mass specifically and that can induce immunological memory to control tumor relapse. A lot of research focused on the injection of DC vaccines in patients, but in vivo DC targeting strategies and the use of combinational therapies and adjuvants are now emerging as strategies for the development of new generation DC vaccines.
DC-based immunotherapy
Most DC vaccines consist of DCs or monocyte precursors of DCs that are isolated from the patient, loaded ex vivo with tumor-derived antigens, matured and administered back to the patient. These DC-based vaccines have proven to be safe
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Figure 3. Molecular pathways leading to cross-presentation in DCs. DCs take up antigen by three different mechanisms: receptor-mediated endocytosis, phagocytosis or macropinocytosis. Once the antigen reaches the endolysosomal pathway, depending on the specific routing, it may be degraded by the concourse of the mild pH and different types of cathepsins and other proteases. At this point, properly degraded antigen can be directly loaded into recycling MHC-I in the phagosome (vacuolar pathway). Antigen that still needs further processing must be transported to the cytosol (cytosolic pathway) wehre it is degraded, together with endogenous proteins and DRiPs, by the proteasome. These peptides are transported by TAP or a yet uncharacterized transporter into the ER where they are loaded into MHC-I with the help of the peptide- loading complex. Further trimming in the ER prior to loading is possible by the presence of ER-localized endopeptidases (ERAP1 and 2). R, ribosome; CNX, calnexin; CRT, calreticulin; b2m, b2microglobulin; UGT1, UDP-glucose:glycoprotein glucosultransferase 1; ERAP1/2, ER-aminopeptidases1/2; PLC, peptide-loading complex; Erp57, protein disulfide isomerase 3; TAP1/2, transporter associated with antigen presentation 1/2; DRiPs, defective ribosomal products; ROS, reactive oxygen species; NOX2, NADPH oxidase 2; CLR, C-type lectin receptor; FcR, Fc receptor ; SR, scavenger receptor.
Golgi
ER
R
TAP2
R
MHC-I CRT
β2m Tapasin
ERp57 TAP1
ERAP1 PLC ERAP2
DRiPs
Endogenous (glyco)proteins
Exogenous proteins Protein Synthesis
UGT1
CNX
Proteasome
Phagosome
??
Phagocytosis Macropinocytosis
CLR FcR SR
Receptor-mediated endocytosis
Late endosome Lysosome
Early endosome
Cathepsins
Proteases NOX2
ROS
ROS NOX2
Vacuolar pathway
Cytosolic pathway
?
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and induced the expansion of circulating CD4+ and CD8+ T cells that are specific for tumor antigens[68]. In addition, an overall clinical benefit rate of 30-54% has been shown in advanced melanoma, prostate cancer and renal cell cancer[69;70].
However, ex vivo generated DC vaccines show some disadvantages, like poor DC migration after administration in the patient and the logistics and costs associated with in vitro-manufactured, personalized vaccines[71]. So, therapies targeting standardized tumor vaccines to CLRs expressed by DCs in vivo are currently designed to circumvent these issues, to allow specific targeting of single or multiple DC subsets in vivo. As described earlier, human skin contains a high number of specialized DC subsets that can rapidly migrate, mature and induce immune responses, making the skin a preferred site for the administration of DC-targeted vaccines.
CLRs as targeting receptors for anti-tumor vaccines
Many different CLRs have been used to target anti-tumor vaccines to DCs and induce T cell responses. Of those, DEC-205 targeting is mostly studied and also recently tested in a phase I trial using NY-ESO-1 protein conjugated to DEC-205 targeting antibodies[72]. Earlier studies have already been demonstrated that DEC-205 targeting resulted in antigen internalization, processing and the induction of Th1 and CD8+ T cell responses[73-75]. In addition, induction of T cell responses can be achieved when antigens are targeted to other CLRs expressed by various murine and human DC subsets, such as antigen targeting to DC-SIGN[29;41;43;44;76], langerin[77], DCIR[78] and DNGR-1/CLEC9a[29;41;43;44;79;80]. In particular, DC- SIGN and langerin, which are highly expressed on distinct human skin DC subsets, are primarily investigated in this thesis.
Adjuvants
In general, DC maturation enhances the potency of DC to cross-present antigens.
DC maturation is one of the results of an adjuvant. Adjuvants serve to enhance the magnitude, quality and longevity of specific immune responses to antigens, but have minimal toxicity or lasting immune effects on their own[81]. Especially in the case of cancer immunotherapy adjuvants are of importance, since most therapies are aimed to induce immune responses against relatively weak self-derived antigens and occur in an immune suppressive environment due to the presence of tumor- derived factors[82]. A large set of TLR ligands are known that act as adjuvants and stimulate cross-presentation. Because each DC subset might express a specific set of TLR receptors, they may differently respond to TLR ligands, thereby influencing cross-presentation and therapy efficiency. Adjuvants also have been shown to influence the outcome of CLR-targeted immunotherapies. For instance, in the absence of a maturation stimulus antigen-targeting to DEC-205 resulted in peripheral CD8+ T cell tolerance, while strong in vivo immune responses were observed when DEC-205 targeted antigen was simultaneously delivered with agonistic CD40 antibody as adjuvant[83].
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Currently, polyinosinic:polycytidylic acid (polyI:C) and its derivative poly-ICLC are extensively studied in human patients as cancer vaccine adjuvant. Poly-ICLC adjuvanted DC vaccines has proven to be safe and well-tolerated, induced objective immunological responses, such as a skewing towards a Th1 profile and functional CD8+ and CD4+ T cells and enhanced clinical efficacy of the cancer vaccines[84].
Besides polyI:C, also administration of the cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF), creams containing imiquimod or resiquimod and CD40 are studied in human patients as adjuvants for cancer vaccines. GM-CSF has been shown to induce DC differentiation and maturation, resulting in enhanced expression of co-stimulatory molecules such as CD80 and CD86 and secretion of the pro-inflammatory cytokines TNF-α, IL6 and IL-12[85]. In addition, GM-CSF prolonged the survival of neutrophils and eosinophils and mobilized myeloid cells into the blood[85]. Although initial, uncontrolled clinical trials reported immunological responses and a correlation with clinical outcome after co-administration of GM- CSF and a multipeptide melanoma vaccine (including gp100 and tyrosinase peptides) [86-88], recent results from clinical studies evaluating GM-CSF as an adjuvant to melanoma vaccines suggest that the biologic effects of GM-CSF are complex and can be influenced by multiple factors[89].
In addition, Aldara, an FDA-approved immune response modifier skin cream, containing 5% of the TLR7 agonist imiquimod is also studied as therapy for skin lesion. Aldara is mostly used to treat non-melanoma skin tumors. Recently it was shown that application of Aldara cream results in inflammasome activation and IL-1 release by keratinocytes in naïve murine skin[90]. This effect was mediated independent of TLR7 activation and attributed to isostearic acid, the major component of the vehicle. However, for induction of full inflammation, both imiquimod and the vehicle cream were shown to be required. Following topical application of Aldara skin cream to human skin explant, we observed enhanced migration and maturation of dermal DCs[91]. Combining the Aldara skin cream with MART-1-peptide vaccination in human skin affected the migratory potential of CD14+ skin DC, which was associated with up-regulation of co-stimulatory molecules and increased activation and IFN-γ secretion of MART-1-specific CD8+ T cells.
Notably, the enhanced effects on DC and T cell activity were not observed when injecting soluble TLR7 and/or 8 ligands intradermally. Altogether, these results demonstrate the importance of the combination of antigen and adjuvants used to evoke antigen-specific immune responses.
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Thesis outline
In this thesis, we explored the potential of glycan-modification of melanoma antigens (gp100 and MART-1) as anti-tumor vaccine for the in situ targeting of human skin APC subsets to induce gp100- or MART-1-specific CD8+ and CD4+ T cell responses. In chapter 2 we describe the phenotypical and functional properties of the main skin DC subsets after isolation and migration from human skin. The capacity of the human skin DC subsets to respond to glycan-modified antigens was investigated in the following chapters. In chapter 3, we focused on the induction of MART-1-specific CD4+ and CD8+ T cell responses by CD14+ dDCs after targeting the receptor DC-SIGN using glycan-modified liposomes containing MART-1 peptides.
We found that targeting of DC-SIGN using glycan-modified liposomes greatly enhances the cross-presentation capacity of migrated CD14+ dDCs, especially when liposomes were simultaneously injected with the cytokines GM-CSF and IL-4. These cytokines improved DC migration from the skin and induced higher expression of DC-SIGN on both the CD1a+ and CD14+ dDCs. In contrast to the dDCs, we observed that human LCs cannot be targeted using glycan-modified liposomes. However, chapter 4 provides evidence that LC targeting can be achieved using glycan-modified peptides that target to the LC-specific receptor langerin and enhance induction of CD4+ and CD8+ T cell responses. The different requirements between langerin and DC-SIGN for targeting are further investigated in chapter 5. Here we demonstrate that both DC-SIGN and langerin recognize the glycan LewisY (LeY), which allows comparison of targeting between both receptors using the same glycan. However, we found that DC-SIGN+ DCs can be successfully targeted using multivalent LeY- modified liposomes with a diameter of approximately 200 nm, resulting in tumor- specific T cell responses, but no significant results were observed when DC-SIGN was targeted using univalent LeY- modified antigenic peptides of various lengths. On the other hand, targeting of langerin with LeY- modified antigenic peptides lead to langerin-mediated internalization and enhanced T cell responses. In contrast, langerin targeting using the multivalent LeY-modified liposomes did not facilitate langerin internalization and induction of T cell responses, providing evidence that langerin and DC-SIGN, and LCs and DCs respectively, require different sized vaccine formulations for the induction of anti-tumor immune responses. In chapter 6 we investigated a panel of adjuvants on their capacity to induce superior DC migration, maturation and T cell responses in combination with intradermal peptide vaccination. Surprisingly, we have shown that the TLR7 ligand imiquimod-containing cream Aldara induced superior DC migration, maturation and subsequent T cell activation compared to intradermal injection of various soluble TLR ligands. Finally, the obtained results are integrated in a general discussion, presented in chapter 7.
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References
1. Nestle,F.O., Di,M.P., Qin,J.Z., and Nickoloff,B.J., Skin immune sentinels in health and disease.
Nat.Rev.Immunol. 2009. 9: 679-691.
2. Pasparakis,M., Haase,I., and Nestle,F.O., Mechanisms regulating skin immunity and inflammation.
Nat.Rev.Immunol. 2014. 14: 289-301.
3. Nestle,F.O., Zheng,X.G., Thompson,C.B., Turka,L.A., and Nickoloff,B.J., Characterization of dermal dendritic cells obtained from normal human skin reveals phenotypic and functionally distinctive subsets. J.Immunol. 1993. 151: 6535-6545.
4. Geissmann,F., Manz,M.G., Jung,S., Sieweke,M.H., Merad,M., and Ley,K., Development of monocytes, macrophages, and dendritic cells. Science 2010. 327: 656-661.
5. Merad,M., Ginhoux,F., and Collin,M., Origin, homeostasis and function of Langerhans cells and other langerin-expressing dendritic cells. Nat.Rev.Immunol. 2008. 8: 935-947.
6. Klechevsky,E., Morita,R., Liu,M., Cao,Y., Coquery,S., Thompson-Snipes,L., Briere,F.et al., Functional specializations of human epidermal Langerhans cells and CD14+ dermal dendritic cells. Immunity. 2008. 29: 497-510.
7. Banchereau,J., Thompson-Snipes,L., Zurawski,S., Blanck,J.P., Cao,Y., Clayton,S., Gorvel,J.P.et al., The differential production of cytokines by human Langerhans cells and dermal CD14(+) DCs controls CTL priming. Blood 2012. 119: 5742-5749.
8. Seneschal,J., Clark,R.A., Gehad,A., Baecher-Allan,C.M., and Kupper,T.S., Human epidermal Langerhans cells maintain immune homeostasis in skin by activating skin resident regulatory T cells. Immunity. 2012. 36: 873-884.
9. McGovern,N., Schlitzer,A., Gunawan,M., Jardine,L., Shin,A., Poyner,E., Green,K.et al., Human Dermal CD14(+) Cells Are a Transient Population of Monocyte-Derived Macrophages. Immunity.
2014. 41: 465-477.
10. Segura,E., Valladeau-Guilemond,J., Donnadieu,M.H., Sastre-Garau,X., Soumelis,V., and Amigorena,S., Characterization of resident and migratory dendritic cells in human lymph nodes.
J.Exp.Med. 2012. 209: 653-660.
11. Sen,D., Forrest,L., Kepler,T.B., Parker,I., and Cahalan,M.D., Selective and site-specific mobilization of dermal dendritic cells and Langerhans cells by Th1- and Th2-polarizing adjuvants. Proc.Natl.
Acad.Sci.U.S.A 2010. 107: 8334-8339.
12. Mathers,A.R. and Larregina,A.T., Professional antigen-presenting cells of the skin. Immunol.Res.
2006. 36: 127-136.
13. Mathers,A.R., Janelsins,B.M., Rubin,J.P., Tkacheva,O.A., Shufesky,W.J., Watkins,S.C., Morelli,A.E.et al., Differential capability of human cutaneous dendritic cell subsets to initiate Th17 responses. J.Immunol. 2009. 182: 921-933.
14. Morelli,A.E., Rubin,J.P., Erdos,G., Tkacheva,O.A., Mathers,A.R., Zahorchak,A.F., Thomson,A.W.et al., CD4+ T cell responses elicited by different subsets of human skin migratory dendritic cells.
J.Immunol. 2005. 175: 7905-7915.
15. Haniffa,M., Shin,A., Bigley,V., McGovern,N., Teo,P., See,P., Wasan,P.S.et al., Human tissues contain CD141hi cross-presenting dendritic cells with functional homology to mouse CD103+
nonlymphoid dendritic cells. Immunity. 2012. 37: 60-73.
16. Chu,C.C., Ali,N., Karagiannis,P., Di,M.P., Skowera,A., Napolitano,L., Barinaga,G.et al., Resident CD141 (BDCA3)+ dendritic cells in human skin produce IL-10 and induce regulatory T cells that suppress skin inflammation. J.Exp.Med. 2012. 209: 935-945.
17. van de Ven,R., van den Hout,M.F., Lindenberg,J.J., Sluijter,B.J., van Leeuwen,P.A., Lougheed,S.M., Meijer,S.et al., Characterization of four conventional dendritic cell subsets in human skin-draining lymph nodes in relation to T-cell activation. Blood 2011. 118: 2502-2510.
18. Kawasaki,T. and Kawai,T., Toll-like receptor signaling pathways. Front Immunol. 2014. 5: 461.
19. Iwasaki,A. and Medzhitov,R., Regulation of adaptive immunity by the innate immune system.
Science 2010. 327: 291-295.
1
20. Kawai,T. and Akira,S., Toll-like receptors and their crosstalk with other innate receptors in infection and immunity. Immunity. 2011. 34: 637-650.
21. Geijtenbeek,T.B., van Vliet,S.J., Engering,A., ‘t Hart,B.A., and van Kooyk,Y., Self- and nonself- recognition by C-type lectins on dendritic cells. Annu.Rev.Immunol. 2004. 22: 33-54.
22. Figdor,C.G., van Kooyk,Y. and Adema,G.J., C-type lectin receptors on dendritic cells and Langerhans cells. Nat.Rev.Immunol. 2002. 2: 77-84.
23. Zelensky,A.N. and Gready,J.E., The C-type lectin-like domain superfamily. FEBS J. 2005. 272:
6179-6217.
24. Geijtenbeek,T.B., Torensma,R., van Vliet,S.J., van Duijnhoven,G.C., Adema,G.J., van Kooyk,Y.
and Figdor,C.G., Identification of DC-SIGN, a novel dendritic cell-specific ICAM-3 receptor that supports primary immune responses. Cell 2000. 100: 575-585.
25. Valladeau,J., Ravel,O., zutter-Dambuyant,C., Moore,K., Kleijmeer,M., Liu,Y., Duvert-Frances,V.et al., Langerin, a novel C-type lectin specific to Langerhans cells, is an endocytic receptor that induces the formation of Birbeck granules. Immunity. 2000. 12: 71-81.
26. van Kooyk,Y. and Geijtenbeek,T.B., DC-SIGN: escape mechanism for pathogens. Nat.Rev.Immunol.
2003. 3: 697-709.
27. Tateno,H., Ohnishi,K., Yabe,R., Hayatsu,N., Sato,T., Takeya,M., Narimatsu,H.et al., Dual specificity of Langerin to sulfated and mannosylated glycans via a single C-type carbohydrate recognition domain. J.Biol.Chem. 2010. 285: 6390-6400.
28. Appelmelk,B.J., van Die,I., van Vliet,S.J., Vandenbroucke-Grauls,C.M., Geijtenbeek,T.B., and van Kooyk,Y., Cutting edge: carbohydrate profiling identifies new pathogens that interact with dendritic cell-specific ICAM-3-grabbing nonintegrin on dendritic cells. J.Immunol. 2003. 170:
1635-1639.
29. Andreini,M., Doknic,D., Sutkeviciute,I., Reina,J.J., Duan,J., Chabrol,E., Thepaut,M.et al., Second generation of fucose-based DC-SIGN ligands: affinity improvement and specificity versus Langerin.
Org.Biomol.Chem. 2011. 9: 5778-5786.
30. Lee,R.T., Hsu,T.L., Huang,S.K., Hsieh,S.L., Wong,C.H., and Lee,Y.C., Survey of immune-related, mannose/fucose-binding C-type lectin receptors reveals widely divergent sugar-binding specificities. Glycobiology 2011. 21: 512-520.
31. van Liempt,E., Bank,C.M., Mehta,P., Garcia-Vallejo,J.J., Kawar,Z.S., Geyer,R., Alvarez,R.A.et al., Specificity of DC-SIGN for mannose- and fucose-containing glycans. FEBS Lett. 2006. 580: 6123- 6131.
32. Mitchell,D.A., Fadden,A.J., and Drickamer,K., A novel mechanism of carbohydrate recognition by the C-type lectins DC-SIGN and DC-SIGNR. Subunit organization and binding to multivalent ligands. J.Biol.Chem. 2001. 276: 28939-28945.
33. Feinberg,H., Mitchell,D.A., Drickamer,K., and Weis,W.I., Structural basis for selective recognition of oligosaccharides by DC-SIGN and DC-SIGNR. Science 2001. 294: 2163-2166.
34. Feinberg,H., Guo,Y., Mitchell,D.A., Drickamer,K., and Weis,W.I., Extended neck regions stabilize tetramers of the receptors DC-SIGN and DC-SIGNR. J.Biol.Chem. 2005. 280: 1327-1335.
35. Cambi,A., de Lange,F., van Maarseveen,N.M., Nijhuis,M., Joosten,B., van Dijk,E.M., de Bakker,B.I.et al., Microdomains of the C-type lectin DC-SIGN are portals for virus entry into dendritic cells. J.Cell Biol. 2004. 164: 145-155.
36. Geijtenbeek,T.B., Kwon,D.S., Torensma,R., van Vliet,S.J., van Duijnhoven,G.C., Middel,J., Cornelissen,I.L.et al., DC-SIGN, a dendritic cell-specific HIV-1-binding protein that enhances trans-infection of T cells. Cell 2000. 100: 587-597.
37. Cambi,A., Gijzen,K., de Vries,l., Torensma,R., Joosten,B., Adema,G.J., Netea,M.G.et al., The C-type lectin DC-SIGN (CD209) is an antigen-uptake receptor for Candida albicans on dendritic cells. Eur.J.Immunol. 2003. 33: 532-538.
38. Pohlmann,S., Zhang,J., Baribaud,F., Chen,Z., Leslie,G.J., Lin,G., Granelli-Piperno,A.et al., Hepatitis C virus glycoproteins interact with DC-SIGN and DC-SIGNR. J.Virol. 2003. 77: 4070-4080.
39. Lozach,P.Y., Lortat-Jacob,H., de Lacroix de,L.A., Staropoli,I., Foung,S., Amara,A., Houles,C.et al.,
1
DC-SIGN and L-SIGN are high affinity binding receptors for hepatitis C virus glycoprotein E2. J.Biol.
Chem. 2003. 278: 20358-20366.
40. Geijtenbeek,T.B., van Vliet,S.J., Koppel,E.A., Sanchez-Hernandez,M., Vandenbroucke- Grauls,C.M., Appelmelk,B., and van Kooyk,Y., Mycobacteria target DC-SIGN to suppress dendritic cell function. J.Exp.Med. 2003. 197: 7-17.
41. Kretz-Rommel,A., Qin,F., Dakappagari,N., Torensma,R., Faas,S., Wu,D., and Bowdish,K.S., In vivo targeting of antigens to human dendritic cells through DC-SIGN elicits stimulatory immune responses and inhibits tumor growth in grafted mouse models. J.Immunother. 2007. 30: 715-726.
42. Tacken,P.J., de Vries,I., Gijzen,K., Joosten,B., Wu,D., Rother,R.P., Faas,S.J.et al., Effective induction of naive and recall T-cell responses by targeting antigen to human dendritic cells via a humanized anti-DC-SIGN antibody. Blood 2005. 106: 1278-1285.
43. Singh,S.K., Stephani,J., Schaefer,M., Kalay,H., Garcia-Vallejo,J.J., den Haan,J., Saeland,E.et al., Targeting glycan modified OVA to murine DC-SIGN transgenic dendritic cells enhances MHC class I and II presentation. Mol.Immunol. 2009. 47: 164-174.
44. Unger,W.W., van Beelen,A.J., Bruijns,S.C., Joshi,M., Fehres,C.M., van Bloois,L., Verstege,M.I.et al., Glycan-modified liposomes boost CD4+ and CD8+ T-cell responses by targeting DC-SIGN on dendritic cells. J.Control Release 2012. 160: 88-95.
45. McDermott,R., Ziylan,U., Spehner,D., Bausinger,H., Lipsker,D., Mommaas,M., Cazenave,J.P.et al., Birbeck granules are subdomains of endosomal recycling compartment in human epidermal Langerhans cells, which form where Langerin accumulates. Mol.Biol.Cell 2002. 13: 317-335.
46. Thepaut,M., Valladeau,J., Nurisso,A., Kahn,R., Arnou,B., Vives,C., Saeland,S.et al., Structural studies of langerin and Birbeck granule: a macromolecular organization model. Biochemistry 2009. 48: 2684-2698.
47. de Witte,L., Nabatov,A., Pion,M., Fluitsma,D., de Jong,M.A., de Gruijl,T., Piguet,V.et al., Langerin is a natural barrier to HIV-1 transmission by Langerhans cells. Nat.Med. 2007. 13: 367-371.
48. Van der Vlist,M., deWitte,L., de Vries,R.D., Litjens,M., de Jong,M.A., Fluitsma,D., de Swart,R.L.et al., Human Langerhans cells capture measles virus through Langerin and present viral antigens to CD4(+) T cells but are incapable of cross-presentation. Eur.J.Immunol. 2011. 41: 2619-2631.
49. de Jong,M.A., Vriend,L.E., Theelen,B., Taylor,M.E., Fluitsma,D., Boekhout,T., and Geijtenbeek,T.B., C-type lectin Langerin is a beta-glucan receptor on human Langerhans cells that recognizes opportunistic and pathogenic fungi. Mol.Immunol. 2010. 47: 1216-1225.
50. Feinberg,H., Powlesland,A.S., Taylor,M.E., and Weis,W.I., Trimeric structure of langerin. J.Biol.
Chem. 2010. 285: 13285-13293.
51. Neefjes,J. and Ovaa,H., A peptide’s perspective on antigen presentation to the immune system.
Nat.Chem.Biol. 2013. 9: 769-775.
52. Rocha,N. and Neefjes,J., MHC class II molecules on the move for successful antigen presentation.
EMBO J. 2008. 27: 1-5.
53. Vyas,J.M., Van der Veen,A.G., and Ploegh,H.L., The known unknowns of antigen processing and presentation. Nat.Rev.Immunol. 2008. 8: 607-618.
54. Blum,J.S., Wearsch,P.A., and Cresswell,P., Pathways of antigen processing. Annu.Rev.Immunol.
2013. 31: 443-473.
55. Yewdell,J.W., DRiPs solidify: progress in understanding endogenous MHC class I antigen processing. Trends Immunol. 2011. 32: 548-558.
56. Colbert,J.D., Farfan-Arribas,D.J., and Rock,K.L., Substrate-induced protein stabilization reveals a predominant contribution from mature proteins to peptides presented on MHC class I. J.Immunol.
2013. 191: 5410-5419.
57. Kamphorst,A.O., Guermonprez,P., Dudziak,D., and Nussenzweig,M.C., Route of antigen uptake differentially impacts presentation by dendritic cells and activated monocytes. J.Immunol. 2010.
185: 3426-3435.
58. Flinsenberg,T.W., Compeer,E.B., Boelens,J.J., and Boes,M., Antigen cross-presentation: extending recent laboratory findings to therapeutic intervention. Clin.Exp.Immunol. 2011. 165: 8-18.
1
59. Kovacsovics-Bankowski,M. and Rock,K.L., A phagosome-to-cytosol pathway for exogenous antigens presented on MHC class I molecules. Science 1995. 267: 243-246.
60. Maupin-Furlow,J., Proteasomes and protein conjugation across domains of life. Nat.Rev.
Microbiol. 2012. 10: 100-111.
61. Macagno,A., Gilliet,M., Sallusto,F., Lanzavecchia,A., Nestle,F.O., and Groettrup,M., Dendritic cells up-regulate immunoproteasomes and the proteasome regulator PA28 during maturation.
Eur.J.Immunol. 1999. 29: 4037-4042.
62. Basler,M., Kirk,C.J., and Groettrup,M., The immunoproteasome in antigen processing and other immunological functions. Curr.Opin.Immunol. 2013. 25: 74-80.
63. Merzougui,N., Kratzer,R., Saveanu,L., and van Endert,P., A proteasome-dependent, TAP- independent pathway for cross-presentation of phagocytosed antigen. EMBO Rep. 2011. 12:
1257-1264.
64. Ramachandra,L., Simmons,D., and Harding,C.V., MHC molecules and microbial antigen processing in phagosomes. Curr.Opin.Immunol. 2009. 21: 98-104.
65. Segura,E. and Villadangos,J.A., A modular and combinatorial view of the antigen cross- presentation pathway in dendritic cells. Traffic. 2011. 12: 1677-1685.
66. Burgdorf,S., Scholz,C., Kautz,A., Tampe,R., and Kurts,C., Spatial and mechanistic separation of cross-presentation and endogenous antigen presentation. Nat.Immunol. 2008. 9: 558-566.
67. Delamarre,L., Pack,M., Chang,H., Mellman,I., and Trombetta,E.S., Differential lysosomal proteolysis in antigen-presenting cells determines antigen fate. Science 2005. 307: 1630-1634.
68. Palucka,K. and Banchereau,J., Cancer immunotherapy via dendritic cells. Nat.Rev.Cancer 2012.
12: 265-277.
69. Engell-Noerregaard,L., Hansen,T.H., Andersen,M.H., Thor,S.P., and Svane,I.M., Review of clinical studies on dendritic cell-based vaccination of patients with malignant melanoma: assessment of correlation between clinical response and vaccine parameters. Cancer Immunol.Immunother.
2009. 58: 1-14.
70. Draube,A., Klein-Gonzalez,N., Mattheus,S., Brillant,C., Hellmich,M., Engert,A., and von Bergwelt-Baildon,M., Dendritic cell based tumor vaccination in prostate and renal cell cancer: a systematic review and meta-analysis. PLoS.One. 2011. 6: e18801.
71. Radford,K.J., Tullett,K.M., and Lahoud,M.H., Dendritic cells and cancer immunotherapy. Curr.
Opin.Immunol. 2014. 27: 26-32.
72. Dhodapkar,M.V., Sznol,M., Zhao,B., Wang,D., Carvajal,R.D., Keohan,M.L., Chuang,E.et al., Induction of antigen-specific immunity with a vaccine targeting NY-ESO-1 to the dendritic cell receptor DEC-205. Sci.Transl.Med. 2014. 6: 232ra51.
73. Sartorius,R., Bettua,C., D’Apice,L., Caivano,A., Trovato,M., Russo,D., Zanoni,I.et al., Vaccination with filamentous bacteriophages targeting DEC-205 induces DC maturation and potent anti- tumor T-cell responses in the absence of adjuvants. Eur.J.Immunol. 2011. 41: 2573-2584.
74. Bonifaz,L.C., Bonnyay,D.P., Charalambous,A., Darguste,D.I., Fujii,S., Soares,H., Brimnes,M.K.et al., In vivo targeting of antigens to maturing dendritic cells via the DEC-205 receptor improves T cell vaccination. J.Exp.Med. 2004. 199: 815-824.
75. Mahnke,K., Guo,M., Lee,S., Sepulveda,H., Swain,S.L., Nussenzweig,M., and Steinman,R.M., The dendritic cell receptor for endocytosis, DEC-205, can recycle and enhance antigen presentation via major histocompatibility complex class II-positive lysosomal compartments. J.Cell Biol. 2000.
151: 673-684.
76. Hesse,C., Ginter,W., Forg,T., Mayer,C.T., Baru,A.M., rnold-Schrauf,C., Unger,W.W.et al., In vivo targeting of human DC-SIGN drastically enhances CD8(+) T-cell-mediated protective immunity.
Eur.J.Immunol. 2013. 43: 2543-2553.
77. Idoyaga,J., Lubkin,A., Fiorese,C., Lahoud,M.H., Caminschi,I., Huang,Y., Rodriguez,A.et al., Comparable T helper 1 (Th1) and CD8 T-cell immunity by targeting HIV gag p24 to CD8 dendritic cells within antibodies to Langerin, DEC205, and Clec9A. Proc.Natl.Acad.Sci.U.S.A 2011. 108:
2384-2389.
1
78. Klechevsky,E., Flamar,A.L., Cao,Y., Blanck,J.P., Liu,M., O’Bar,A., gouna-Deciat,O.et al., Cross- priming CD8+ T cells by targeting antigens to human dendritic cells through DCIR. Blood 2010.
116: 1685-1697.
79. Sancho,D., Mourao-Sa,D., Joffre,O.P., Schulz,O., Rogers,N.C., Pennington,D.J., Carlyle,J.R.et al., Tumor therapy in mice via antigen targeting to a novel, DC-restricted C-type lectin. J.Clin.Invest 2008. 118: 2098-2110.
80. Picco,G., Beatson,R., Taylor-Papadimitriou,J., and Burchell,J.M., Targeting DNGR-1 (CLEC9A) with antibody/MUC1 peptide conjugates as a vaccine for carcinomas. Eur.J.Immunol. 2014. 44:
1947-1955.
81. Dubensky,T.W., Jr. and Reed,S.G., Adjuvants for cancer vaccines. Semin.Immunol. 2010. 22: 155- 82. Finn,O.J., Cancer immunology. N.Engl.J.Med. 2008. 358: 2704-2715.161.
83. Bonifaz,L., Bonnyay,D., Mahnke,K., Rivera,M., Nussenzweig,M.C., and Steinman,R.M., Efficient targeting of protein antigen to the dendritic cell receptor DEC-205 in the steady state leads to antigen presentation on major histocompatibility complex class I products and peripheral CD8+ T cell tolerance. J.Exp.Med. 2002. 196: 1627-1638.
84. Ammi,R., De Waele,J., Willemen,Y., Van Brussel,I., Schrijvers,D.M., Lion,E., and Smits,E.L., Poly(I:C) as cancer vaccine adjuvant: Knocking on the door of medical breakthroughs. Pharmacol.
Ther. 2014.
85. Metcalf,D., Hematopoietic cytokines. Blood 2008. 111: 485-491.
86. Slingluff,C.L., Jr., Petroni,G.R., Yamshchikov,G.V., Barnd,D.L., Eastham,S., Galavotti,H., Patterson,J.W.et al., Clinical and immunologic results of a randomized phase II trial of vaccination using four melanoma peptides either administered in granulocyte-macrophage colony-stimulating factor in adjuvant or pulsed on dendritic cells. J.Clin.Oncol. 2003. 21: 4016-4026.
87. Chianese-Bullock,K.A., Pressley,J., Garbee,C., Hibbitts,S., Murphy,C., Yamshchikov,G., Petroni,G.R.et al., MAGE-A1-, MAGE-A10-, and gp100-derived peptides are immunogenic when combined with granulocyte-macrophage colony-stimulating factor and montanide ISA-51 adjuvant and administered as part of a multipeptide vaccine for melanoma. J.Immunol. 2005.
174: 3080-3086.
88. Jager,E., Ringhoffer,M., Dienes,H.P., Arand,M., Karbach,J., Jager,D., Ilsemann,C.et al., Granulocyte-macrophage-colony-stimulating factor enhances immune responses to melanoma- associated peptides in vivo. Int.J.Cancer 1996. 67: 54-62.
89. Kaufman,H.L., Ruby,C.E., Hughes,T., and Slingluff,C.L., Jr., Current status of granulocyte- macrophage colony-stimulating factor in the immunotherapy of melanoma. J.Immunother.Cancer 2014. 2: 11.
90. Walter,A., Schafer,M., Cecconi,V., Matter,C., Urosevic-Maiwald,M., Belloni,B., Schonewolf,N.et al., Aldara activates TLR7-independent immune defence. Nat.Commun. 2013. 4: 1560.
91. Fehres,C.M., Bruijns,S.C., van Beelen,A.J., Kalay,H., Ambrosini,M., Hooijberg,E., Unger,W.W.et al., Topical rather than intradermal application of the TLR7 ligand imiquimod leads to human dermal dendritic cell maturation and CD8+ T-cell cross-priming. Eur.J.Immunol. 2014. 44: 2415- 2424.
