Paul J. Tacken

Dendritic-cell immunotherapy: from ex vivo loading to in vivo targeting.

The realization that dendritic cells (DCs) orchestrate innate and adaptive immune responses has stimulated research on harnessing DCs to create more effective vaccines. Early clinical trials exploring autologous DCs that were loaded with antigens ex vivo to induce T-cell responses have provided proof of principle. Here, we discuss how direct targeting of antigens to DC surface receptors in vivo might replace laborious and expensive ex vivo culturing, and facilitate large-scale application of DC-based vaccination therapies.

Targeting DCIR on human plasmacytoid dendritic cells results in antigen presentation and inhibits IFN-alpha production.

C-type lectin receptors (CLRs) fulfill multiple functions within the immune system by recognition of carbohydrate moieties on foreign or (altered) self-structures. CLRs on myeloid dendritic cells (DCs) have been well characterized as pattern-recognition receptors (PRRs) combining ligand internalization with complex signaling events. Much less is known about CLR expression and function in human plasmacytoid DCs (pDCs), the major type I interferon (IFN) producers. In this study, we demonstrate that, next to the CLR BDCA-2, human pDCs express DC immunoreceptor (DCIR), a CLR with putative immune-inhibitory function, but not dectin-1, mannose receptor, or DC-specific ICAM-3-grabbing nonintegrin. DCIR surface levels are reduced on pDC maturation after TLR9 triggering. Interestingly, DCIR triggering inhibits TLR9-induced IFN-alpha production while leaving up-regulation of costimulatory molecule expression unaffected. Furthermore, DCIR is readily internalized into pDCs after receptor triggering. We show that DCIR internalization is clathrin-dependent because it can be inhibited by hypertonic shock and dominant-negative dynamin. Importantly, antigens targeted to pDCs via DCIR are presented to T cells. These findings indicate that targeting DCIR on pDCs not only results in efficient antigen presentation but also affects TLR9-induced IFN-alpha production. Collectively, the data show that targeting of DCIR can modulate human pDC function and may be applied in disease prevention and treatment.

Targeted PLGA nano- but not microparticles specifically deliver antigen to human dendritic cells via DC-SIGN in vitro.

Vaccine efficacy is strongly enhanced by antibody-mediated targeting of vaccine components to dendritic cells (DCs), which are professional antigen presenting cells. However, the options to link antigens or immune modulators to a single antibody are limited. Here, we engineered versatile nano- and micrometer-sized slow-release vaccine delivery vehicles that specifically target human DCs to overcome this limitation. The nano- (NPs) and microparticles (MPs), with diameters of approximately 200nm and 2microm, consist of a PLGA core coated with a polyethylene glycol-lipid layer carrying the humanized targeting antibody hD1, which does not interact with complement or Fc receptors and recognizes the human C-type lectin receptor DC-SIGN on DCs. We studied how these particles interact with human DCs and blood cells, as well as the kinetics of PLGA-encapsulated antigen degradation within DCs. Encapsulation of antigen resulted in almost 38% degradation for both NPs and MPs 6days after particle ingestion by DCs, compared to 94% when nonencapsulated, soluble antigen was used. In contrast to the MPs, which were taken up rather nonspecifically, the NPs effectively targeted human DCs. Consequently, targeted delivery only improved antigen presentation of NPs and induced antigen-dependent T cell responses at 10-100 fold lower concentrations than nontargeted NPs.

The C type lectin receptor CLEC9A mediates antigen uptake and (cross-)presentation by human blood BDCA3+ myeloid dendritic cells

CLEC9A is a recently discovered C-type lectin receptor involved in sensing necrotic cells. In humans, this receptor is selectively expressed by BDCA3(+) myeloid dendritic cells (mDCs), which have been proposed to be the main human cross-presenting mDCs and may represent the human homologue of murine CD8(+) DCs. In mice, it was demonstrated that antigens delivered with antibodies to CLEC9A are presented by CD8(+) DCs to both CD4(+) and CD8(+) T cells and induce antitumor immunity in a melanoma model. Here we assessed the ability of CLEC9A to mediate antigen presentation by human BDCA3(+) mDCs, which represent < 0.05% of peripheral blood leukocytes. We demonstrate that CLEC9A is only expressed on immature BDCA3(+) mDCs and that cell surface expression is lost after TLR-mediated maturation. CLEC9A triggering via antibody binding rapidly induces receptor internalization but does not affect TLR-induced cytokine production or expression of costimulatory molecules. More importantly, antigens delivered via CLEC9A antibodies to BDCA3(+) mDCs are presented by both MHC class I (cross-presentation) and MHC class II to antigen-specific T cells. We conclude that CLEC9A is a promising target for in vivo antigen delivery in humans to increase the efficiency of vaccines against infectious or malignant diseases.

Targeted delivery of TLR ligands to human and mouse dendritic cells strongly enhances adjuvanticity.

Effective vaccines consist of 2 components: immunodominant antigens and effective adjuvants. Whereas it has been demonstrated that targeted delivery of antigens to dendritic cells (DCs) improves vaccine efficacy, we report here that co-targeting of TLR ligands (TLRLs) to DCs strongly enhances adjuvanticity and immunity. We encapsulated ligands for intracellular TLRs within biodegradable nanoparticles coated with Abs recognizing DC-specific receptors. Targeted delivery of TLRLs to human DCs enhanced the maturation and production of immune stimulatory cytokines and the Ag-specific activation of naive CD8(+) T cells. In vivo studies demonstrated that nanoparticles carrying Ag induced cytotoxic T-lymphocyte responses at 100-fold lower adjuvant dose when TLRLs were co-encapsulated instead of administered in soluble form. Moreover, the efficacy of these targeted TLRLs reduced the serum cytokine storm and related toxicity that is associated with administration of soluble TLRLs. We conclude that the targeted delivery of adjuvants may improve the efficacy and safety of DC-based vaccines.

Controlled release of antigen and Toll-like receptor ligands from PLGA nanoparticles enhances immunogenicity

AIM Dendritic cells rapidly capture nanoparticles and induce a potent cellular immune response. It is yet unknown whether the immunological response induced by slow release of encapsulated versus soluble antigen and adjuvant is superior. MATERIALS & METHODS The kinetics of poly(lactic-co-glycolic acid) PLGA nanoparticles antigen release was studied by the DQ-bovine serum albumin (BSA) self-quenching antigen model. The immunological response induced was evaluated by means of dendritic cell activation/maturation markers, cytokine production and their ability to drive antigen-specific T-cell proliferation. RESULTS & CONCLUSION PLGA-encapsulated antigen and adjuvant showed an enhanced T-cell response when compared with soluble vaccine components by increasing antigenicity and adjuvanticity. Although the kinetic profile followed the same pattern, encapsulation increased strength and duration of the response.

The influence of PEG chain length and targeting moiety on antibody-mediated delivery of nanoparticle vaccines to human dendritic cells

Targeted delivery of nanoparticles (NPs) carrying vaccine components to dendritic cells (DCs) is a promising strategy to initiate antigen-specific immune responses. Improving the interactions between nanoparticle-carried ligands and receptors on DCs is a major challenge. These NPs are generally coated with poly(ethylene glycol) (PEG), to shield non-specific interactions, and antibodies, to facilitate specific delivery to DC surface receptors. We have devised a strategy to covalently link PEG molecules of various chain length (Mw 2000-20000 g/moL) to poly(lactic-co-)glycolic acid (PLGA) NP vaccines. We coated these NPs with various antibodies recognizing the DC-specific receptor DC-SIGN to study the effects of shielding and antibody type on antibody--receptor interactions. Chemical attachment of PEG to the particle surface was followed by detailed zeta potential, DLS and NMR studies, and analyzed by analytical chemistry. Increasing the PEG chain length increased particle size and polydispersity index and reduced the intracellular degradation rate of encapsulated antigens. Binding and uptake of NPs by human DCs was affected by both PEG chain length and antibody type. NPs coated with PEG-3000 had the optimal chain length for antibody--receptor interactions and induction of antigen-specific T-cell responses. Interestingly, clear differences were observed upon targeting distinct epitopes of the same receptor. Binding and uptake of NPs carrying antibodies recognizing the carbohydrate recognition domain of DC-SIGN was enhanced when compared to those carrying antibodies recognizing the receptors neck region. In conclusion, our data show that PEG chains cannot be extended beyond a certain length for shielding purposes without compromising the efficacy of targeted delivery. Thereby, the implications of our findings are not limited to the future design of nanovaccines specifically targeted to DC-SIGN, but apply to the general design of targeted nanocarriers.

Targeted antigen delivery and activation of dendritic cells in vivo: steps towards cost effective vaccines.

During the past decade, the immunotherapeutic potential of ex vivo generated professional antigen presenting dendritic cells (DCs) has been explored in the clinic. Albeit safe, clinical results have thus far been limited. A major disadvantage of current cell-based dendritic cell (DC) therapies, preventing universal implementation of this form of immunotherapy, is the requirement that vaccines need to be tailor made for each individual. Targeted delivery of antigens to DC surface receptors in vivo would circumvent this laborious and expensive ex vivo culturing steps involved with these cell-based therapies. In addition, the opportunity to target natural and often rare DC subsets in vivo might have advantages over loading more artificial ex vivo cultured DCs. Preclinical studies show targeting antigens to DCs effectively induces humoral responses, while cellular responses are induced provided a DC maturation or activation stimulus is co-administered. Here, we discuss strategies to target antigens to distinct DC subsets and to simultaneously employ adjuvants to activate these cells to induce immunity.

Targeting DC-SIGN via its neck region leads to prolonged antigen residence in early endosomes, delayed lysosomal degradation, and cross-presentation.

Targeting antigens to dendritic cell (DC)-specific receptors, such as DC-SIGN, induces potent T cell-mediated immune responses. DC-SIGN is a transmembrane C-type lectin receptor with a long extracellular neck region and a carbohydrate recognition domain (CRD). Thus far, only antibodies binding the CRD have been used to target antigens to DC-SIGN. We evaluated the endocytic pathway triggered by antineck antibodies as well as their intracellular routing and ability to induce CD8(+) T-cell activation. In contrast to anti-CRD antibodies, antineck antibodies induced a clathrin-independent mode of DC-SIGN internalization, as demonstrated by the lack of colocalization with clathrin and the observation that silencing clathrin did not affect antibody internalization in human DCs. Interestingly, we observed that anti-neck and anti-CRD antibodies were differentially routed within DCs. Whereas anti-CRD antibodies were mainly routed to late endosomal compartments, anti-neck antibodies remained associated with early endosomal compartments positive for EEA-1 and MHC class I for up to 2 hours after internalization. Finally, cross-presentation of protein antigen conjugated to antineck antibodies was approximately 1000-fold more effective than nonconjugated antigen. Our studies demonstrate that anti-neck antibodies trigger a distinct mode of DC-SIGN internalization that shows potential for targeted vaccination strategies.

Targeting dendritic cells--why bother?

Vaccination is among the most efficient forms of immunotherapy. Although sometimes inducing lifelong protective B-cell responses, T-cell-mediated immunity remains challenging. Targeting antigen to dendritic cells (DCs) is an extensively explored concept aimed at improving cellular immunity. The identification of various DC subsets with distinct functional characteristics now allows for the fine-tuning of targeting strategies. Although some of these DC subsets are regarded as superior for (cross-) priming of naive T cells, controversies still remain about which subset represents the best target for immunotherapy. Because targeting the antigen alone may not be sufficient to obtain effective T-cell responses, delivery systems have been developed to target multiple vaccine components to DCs. In this Perspective, we discuss the pros and cons of targeting DCs: if targeting is beneficial at all and which vaccine vehicles and immunization routes represent promising strategies to reach and activate DCs.