Dancing with dendritic cells:Targeting human skin dendritic cells for anti-tumor immunity

In the studies described in this thesis we explored the potential of human skin DC to induce anti-tumor immunity. By the design of various vaccines we explored how the multiple human skin DC subsets responded for the induction of robust adaptive anti-tumor immunity. For intradermally injected particulate-vaccines, we need to ensure that the tumor-antigen containing particle will reach the targeted DC subset to facilitate the induction of cytotoxic CD8+ T-cells with tumor cell killing capacity. For this DC need to shuttle exogenously derived antigens into the endogenous presentation pathway, better known as cross-presentation. The expression of DC specific CLR gives us the opportunity to both specifically target skin DC whilst inducing cross-presentatio in combinaiton with TLR activation. In chapter 2 we show Langerin to be superior in the activation of specific CD8+ T-cells. In order to explore whether targeting dermal DC is beneficial in facilitating cross-presentation, we used DC-SIGN as target candidate. In chapter 3 we elucidated the exact intracellular routingof DC-SIGN and its cargo. Furthermore, we show that simultaneous triggering of DC-SIGN using an antibody conjugated to the melanoma specific gp100 SLP and TLR4 using LPS, efficiently enhances cross-presentation. This dual stimulation ensured SLP degradation by the proteasome and antigen processing for MHC I loading. In chapter 4, we used the overlapping glycan binding profiles for the Lewis Y type antigens of Langerin and DC-SIGN to target both receptors. Using Lewis Y (LeY ) as targeting glycan we aimed to generate a single glycovaccine targeting multiple human skin DC subsets simultaneously. As carrier system we used PAMAM-dendrimers, to covalently link the gp100 SLP and create two differentially sized vaccines. We show glycodendrimers of approximately 50nm efficiently target both Langerin and DC-SIGN,thereby reaching multiple human skin DC subsets when injected in situ. Furthermore,this enhanced activation of tumor specific CD4+ and CD8+ T-cells compared to nontargeting dendrimers. In order to investigate whether we could further optimize our cancer vaccine, we investigated whether we could improve DC maturation for cytokine skewing and co-stimulation and antigen processing, by combining various PRR agonists. TLR and NOD-like receptors (NLR) that are expressed by different human skin DC and can synergize for DC maturation and antigen handling. In chapter 5 we used the PAMAMdendrimer, to develop a multivalent antigenic vaccine containing both the gp100 SLP and NOD2 agonist MDP. A synthetic NOD2-agonist, was covalently linked to the multivalent antigenic dendrimer. We show that the combination of the NLR agonist-antigen complex with the soluble TLR4 agonist MPLA enhances cytokine secretion within the skin micromilieu. Furthermore, it enhances cross-presentation by human skin DC for CD8+ T-cell activation. Intradermal vaccine delivery is usually achieved through injection, though efforts are made to design systems that simplify intradermal vaccine delivery. In chapter 6 we made use of an ablative fractional laser to verify whether it might benefit vaccination with our anti-tumor vaccine particles. We show that in our human skin explant model intradermal injection was more efficient for vaccine delivery to and uptake by skin DC, resulting in higher level CD8+ T-cell activation. This thesis aimed to develop a human skin DC targeting cancer vaccine, exploiting the expression by DC of the CLR Langerin and DC-SIGN. By the design of a multivalent glyco-vaccine incorporating melanoma specific gp100 epitopes and the targeting moiety LeY we could efficiently target multiple human skin DC for enhanced (cross)- presentation using a single vaccine formulation. This dual targeting, multivalent vaccine can be used for inclusion of a multitude of epitopes and PRR agonists. Thereby we developed a flexible intradermal vaccine platform which has merit for clinical studies aiming to cure different types of cancer

Dancing with dendritic cells: Targeting human skin dendritic cells for anti-tumor immunity

In the studies described in this thesis we explored the potential of human skin DC to induce anti-tumor immunity. By the design of various vaccines we explored how the multiple human skin DC subsets responded for the induction of robust adaptive anti-tumor immunity. For intradermally injected particulate-vaccines, we need to ensure that the tumor-antigen containing particle will reach the targeted DC subset to facilitate the induction of cytotoxic CD8+ T-cells with tumor cell killing capacity. For this DC need to shuttle exogenously derived antigens into the endogenous presentation pathway, better known as cross-presentation. The expression of DC specific CLR gives us the opportunity to both specifically target skin DC whilst inducing cross-presentatio in combinaiton with TLR activation. In chapter 2 we show Langerin to be superior in the activation of specific CD8+ T-cells. In order to explore whether targeting dermal DC is beneficial in facilitating cross-presentation, we used DC-SIGN as target candidate. In chapter 3 we elucidated the exact intracellular routingof DC-SIGN and its cargo. Furthermore, we show that simultaneous triggering of DC-SIGN using an antibody conjugated to the melanoma specific gp100 SLP and TLR4 using LPS, efficiently enhances cross-presentation. This dual stimulation ensured SLP degradation by the proteasome and antigen processing for MHC I loading. In chapter 4, we used the overlapping glycan binding profiles for the Lewis Y type antigens of Langerin and DC-SIGN to target both receptors. Using Lewis Y (LeY ) as targeting glycan we aimed to generate a single glycovaccine targeting multiple human skin DC subsets simultaneously. As carrier system we used PAMAM-dendrimers, to covalently link the gp100 SLP and create two differentially sized vaccines. We show glycodendrimers of approximately 50nm efficiently target both Langerin and DC-SIGN,thereby reaching multiple human skin DC subsets when injected in situ. Furthermore,this enhanced activation of tumor specific CD4+ and CD8+ T-cells compared to nontargeting dendrimers. In order to investigate whether we could further optimize our cancer vaccine, we investigated whether we could improve DC maturation for cytokine skewing and co-stimulation and antigen processing, by combining various PRR agonists. TLR and NOD-like receptors (NLR) that are expressed by different human skin DC and can synergize for DC maturation and antigen handling. In chapter 5 we used the PAMAMdendrimer, to develop a multivalent antigenic vaccine containing both the gp100 SLP and NOD2 agonist MDP. A synthetic NOD2-agonist, was covalently linked to the multivalent antigenic dendrimer. We show that the combination of the NLR agonist-antigen complex with the soluble TLR4 agonist MPLA enhances cytokine secretion within the skin micromilieu. Furthermore, it enhances cross-presentation by human skin DC for CD8+ T-cell activation. Intradermal vaccine delivery is usually achieved through injection, though efforts are made to design systems that simplify intradermal vaccine delivery. In chapter 6 we made use of an ablative fractional laser to verify whether it might benefit vaccination with our anti-tumor vaccine particles. We show that in our human skin explant model intradermal injection was more efficient for vaccine delivery to and uptake by skin DC, resulting in higher level CD8+ T-cell activation. This thesis aimed to develop a human skin DC targeting cancer vaccine, exploiting the expression by DC of the CLR Langerin and DC-SIGN. By the design of a multivalent glyco-vaccine incorporating melanoma specific gp100 epitopes and the targeting moiety LeY we could efficiently target multiple human skin DC for enhanced (cross)- presentation using a single vaccine formulation. This dual targeting, multivalent vaccine can be used for inclusion of a multitude of epitopes and PRR agonists. Thereby we developed a flexible intradermal vaccine platform which has merit for clinical studies aiming to cure different types of cancer

Glyco-dendrimers as intradermal anti-tumor vaccine targeting multiple skin DC subsets

The human skin is an attractive anti-tumor vaccination site due to the vast network of dendritic cell (DC) subsets that carry antigens to the draining lymph nodes and stimulate tumor specific CD4+ and CD8+ T cells in. Specific vaccine delivery to skin DC can be accomplished by targeting glycan coated antigens to C-type lectin receptors (CLRs) such as DC-SIGN expressed by human dermal DCs and Langerin expressed by Langerhans cells (LCs), which facilitate endocytosis and processing for antigen presentation and T cell activation. Although there are multiple human skin DC subsets, targeting individual DC subsets and receptors has been a focus in the past. However, the simultaneous targeting of multiple human skin DC subsets that mobilize the majority of the skin antigen presenting cells (APC) is preferred to accomplish more robust and efficient T cell stimulation. Dual CLR targeting using a single tumor vaccine has been difficult, as we previously showed Langerin to favor binding and uptake of monovalent glyco-peptides whereas DC-SIGN favors binding of larger multivalent glyco-particles such as glyco-liposomes. Methods: We used branched polyamidoamine (PAMAM) dendrimers as scaffold for melanoma specific gp100 synthetic long peptides and the common DC-SIGN and Langerin ligand Lewis Y (LeY), to create multivalent glyco-dendrimers with varying molecular weights for investigating dual DC-SIGN and Langerin targeting. Using DC-SIGN+ monocyte derived DC (moDC) and Langerin+ primary LC we investigated glyco-dendrimer CLR targeting properties and subsequent gp100 specific CD8+ T cell activation in vitro. In situ targeting ability to human dermal DC and LC through intradermal injection in a human skin explant model was elucidated. Results: Dual DC-SIGN and Langerin binding was achieved using glyco-dendrimers of approximately 100kD, thereby fulfilling our criteria to simultaneously target LCs and CD1a+ and CD14+ dermal DC in situ. Both DC-SIGN and Langerin targeting by glyco-dendrimers resulted in enhanced internalization and gp100 specific CD8+ T cell activation. Conclusion: We designed the first glyco-vaccine with dual CLR targeting properties, thereby reaching multiple human skin DC subsets in situ for improved anti-tumor CD8+ T cell responses

Toll-Like Receptor 4 Triggering Promotes Cytosolic Routing of DC-SIGN-Targeted Antigens for Presentation on MHC Class I

DC-SIGN is an antigen uptake receptor expressed on dendritic cells (DCs) with specificity for glycans present on a broad variety of pathogens and is capable of directing its cargo to MHC-I and MHC-II pathways for the induction of CD8+ and CD4+ T cell responses, respectively. Therefore, DC-SIGN is a very promising target for the delivery of antigen for anti-cancer vaccination. Although the endocytic route leading to MHC-II presentation is characterized to a large extent, the mechanisms controlling DC-SIGN targeted cross-presentation of exogenous peptides on MHC-I, are not completely resolved yet. In this paper, we used imaging flow cytometry and antigen-specific CD8+ T cells to investigate the intracellular fate of DC-SIGN and its cargo in human DCs. Our data demonstrates that immature DCs and toll-like receptor 4 (TLR4) stimulated DCs had similar internalization capacity and were both able to cross-present antigen targeted via DC-SIGN. Interestingly, simultaneous triggering of TLR4 and DC-SIGN on DCs resulted in the translocation of cargo to the cytosol, leading to proteasome-dependent processing and increased CD8+ T cell activation. Understanding the dynamics of DC-SIGN-mediated uptake and processing is essential for the design of optimal DC-SIGN-targeting vaccination strategies aimed at enhancing CD8+ T cell responses

Langerin-mediated internalization of a modified peptide routes antigens to early endosomes and enhances cross-presentation by human Langerhans cells

The potential of the skin immune system to generate immune responses is well established, and the skin is actively exploited as a vaccination site. Human skin contains several antigen-presenting cell subsets with specialized functions. In particular, the capacity to cross-present exogenous antigens to CD8+ T cells is of interest for the design of effective immunotherapies against viruses or cancer. Here, we show that primary human Langerhans cells (LCs) were able to cross-present a synthetic long peptide (SLP) to CD8+T cells. In addition, modification of this SLP using antibodies against the receptor langerin, but not dectin-1, further enhanced the cross-presenting capacity of LCs through routing of internalized antigens to less proteolytic early endosome antigen 1+early endosomes. The potency of LCs to enhance CD8+T-cell responses could be further increased through activation of LCs with the toll-like receptor 3 ligand polyinosinic:polycytidylic acid (pI:C). Altogether, the data provide evidence that human LCs are able to cross-present antigens after langerin-mediated internalization. Furthermore, the potential for antigen modification to target LCs specifically provides a rationale for generating effective anti-tumor or anti-viral cytotoxic T lymphocyte responses

Lipo-Based Vaccines as an Approach to Target Dendritic Cells for Induction of T- and iNKT Cell Responses

In this study we developed a liposome-based vaccine containing palmitoylated synthetic long peptides (SLP) and alpha galactosylceramide (αGC) to specifically target dendritic cells (DC) for activation of both innate (invariant natural killer T-cells [iNKT]) and adaptive (CD8+ T-cells) players of the immune system. Combination of model tumor specific antigens (gp100/MART-1) formulated as a SLP and αGC in one liposome results in strong activation of CD8+ and iNKT, as measured by IFNγ secretion. Moreover, addition of lipo-Lewis Y (LeY) to the liposomes for C-type lectin targeting increased not only uptake by monocyte-derived dendritic cells (moDC), dermal dendritic cells and Langerhans cells but also enhanced gp100-specific CD8+ T- and iNKT cell activation by human skin-emigrated antigen presenting cells in an ex vivo explant model. Loading of moDC with liposomes containing LeY also showed priming of MART-126−35L specific CD8+ T-cells. In conclusion, chemically linking a lipid tail to a glycan-based targeting moiety and SLP combined with αGC in one liposome allows for easy generation of vaccine formulations that target multiple skin DC subsets and induce tumor antigen specific CD8+ T- and iNKT cells. These liposomes present a new vaccination strategy against tumors

Palmitoylated antigens for the induction of anti-tumor CD8+ T cells and enhanced tumor recognition

Induction of tumor-specific cytotoxic CD8+ T cells (CTLs) via immunization relies on the presentation of tumor-associated peptides in major histocompatibility complex (MHC) class I molecules by dendritic cells (DCs). To achieve presentation of exogenous peptides into MHC class I, cytosolic processing and cross-presentation are required. Vaccination strategies aiming to induce tumor-specific CD8+ T cells via this exogenous route therefore pose a challenge. In this study, we describe improved CD8+ T cell induction and in vivo tumor suppression of mono-palmitic acid-modified (C16:0) antigenic peptides, which can be attributed to their unique processing route, efficient receptor-independent integration within lipid bilayers, and continuous intracellular accumulation and presentation through MHC class I. We propose that this membrane-integrating feature of palmitoylated peptides can be exploited as a tool for quick and efficient antigen enrichment and MHC class I loading. Importantly, both DCs and non-professional antigen-presenting cells (APCs), similar to tumor cells, facilitate anti-tumor immunity by efficient CTL priming via DCs and effective recognition of tumors through enhanced presentation of antigens

presentation_1_Toll-Like Receptor 4 Triggering Promotes Cytosolic Routing of DC-SIGN-Targeted Antigens for Presentation on MHC Class I.PDF

<p>DC-SIGN is an antigen uptake receptor expressed on dendritic cells (DCs) with specificity for glycans present on a broad variety of pathogens and is capable of directing its cargo to MHC-I and MHC-II pathways for the induction of CD8⁺ and CD4⁺ T cell responses, respectively. Therefore, DC-SIGN is a very promising target for the delivery of antigen for anti-cancer vaccination. Although the endocytic route leading to MHC-II presentation is characterized to a large extent, the mechanisms controlling DC-SIGN targeted cross-presentation of exogenous peptides on MHC-I, are not completely resolved yet. In this paper, we used imaging flow cytometry and antigen-specific CD8⁺ T cells to investigate the intracellular fate of DC-SIGN and its cargo in human DCs. Our data demonstrates that immature DCs and toll-like receptor 4 (TLR4) stimulated DCs had similar internalization capacity and were both able to cross-present antigen targeted via DC-SIGN. Interestingly, simultaneous triggering of TLR4 and DC-SIGN on DCs resulted in the translocation of cargo to the cytosol, leading to proteasome-dependent processing and increased CD8⁺ T cell activation. Understanding the dynamics of DC-SIGN-mediated uptake and processing is essential for the design of optimal DC-SIGN-targeting vaccination strategies aimed at enhancing CD8⁺ T cell responses.</p

Synergistic effects of dendritic cell targeting and laser-microporation on enhancing epicutaneous skin vaccination efficacy

Due to its unique immunological properties, the skin is an attractive target tissue for allergen-specific immunotherapy. In our current work, we combined a dendritic cell targeting approach with epicutaneous immunization using an ablative fractional laser to generate defined micropores in the upper layers of the skin. By coupling the major birch pollen allergen Bet v 1 to mannan from S. cerevisiae via mild periodate oxidation we generated hypoallergenic Bet-mannan neoglycoconjugates, which efficiently targeted CD14(+) dendritic cells and Langerhans cells in human skin explants. Mannan conjugation resulted in sustained release from the skin and retention in secondary lymphoid organs, whereas unconjugated antigen showed fast renal clearance. In a mouse model, Bet-mannan neoglycoconjugates applied via laser-microporated skin synergistically elicited potent humoral and cellular immune responses, superior to intradermal injection. The induced antibody responses displayed IgE-blocking capacity, highlighting the therapeutic potential of the approach. Moreover, application via micropores, but not by intradermal injection, resulted in a mixed TH1/TH17-biased immune response. Our data clearly show that applying mannan-neoglycoconjugates to an organ rich in dendritic cells using laser-microporation is superior to intradermal injection. Due to their low IgE binding capacity and biodegradability, mannan neoglycoconjugates therefore represent an attractive formulation for allergen-specific epicutaneous immunotherapy