Direct and indirect engagement of dendritic cell function by antibodies developed for cancer therapy.

Dendritic cells (DC) are crucial for the priming of T cells and thereby influence adaptive immune responses. Hence, they also represent important players in shaping anti-tumour immune responses. Cancer immunotherapy has been driven over many years by the aim to harness the T-cell stimulatory activity of these crucial antigen-presenting cells (APC). Efficient antigen delivery alone is not sufficient for full engagement of the T-cell stimulatory activity of DC and the inclusion of adjuvants triggering appropriate DC activation is essential to ensure effective anti-tumour immunity induction. While the direct engagement of DC function is a powerful tool for tumour immunotherapy, many therapeutic antibodies, such as antibodies directed against tumour-associated antigens (TAA) and immune checkpoint inhibitors (ICI) have been shown to engage DC function indirectly. The induction of anti-tumour immune responses by TAA-targeting and immune checkpoint inhibitory antibodies is thought to be integral to their therapeutic efficacy. Here, we provide an overview of the immunotherapeutic antibodies in the context of cancer immunotherapy, that has been demonstrated to directly or indirectly engage DC and discuss the current understanding of the functional mechanisms underlying anti-tumour immunity induction by these antibody therapies. In the future, the combination of therapeutic strategies that engage DC function directly and/or indirectly with strategies that allow tumour infiltrating immune effector cells to exert their anti-tumour activity in the tumour microenvironment (TME) may be key for the successful treatment of cancer patients currently not responding to immunotherapeutic antibody treatment.

Antibody conjugates for targeted delivery of Toll-like receptor 9 agonist to the tumor tissue.

Imiquimod, a Toll-like receptor 7 (TLR7) agonist is routinely used for topical administration in basal cell carcinoma and stage zero melanoma. Similarly, the TLR agonist Bacillus Calmette-Guérin is used for the local treatment of bladder cancer and clinical trials showed treatment efficacy of intratumoral injections with TLR9 agonists. However, when administered systemically, endosomal TLR agonists cause adverse responses due to broad immune activation. Hence, strategies for targeted delivery of TLR agonists to the tumor tissue are needed to enable the widespread use of endosomal TLR agonists in the context of tumor immunotherapy. One strategy for targeted delivery of TLR agonist is their conjugation to tumor antigen-specific therapeutic antibodies. Such antibody-TLR agonist conjugates act synergistically by inducing local TLR-mediated innate immune activation which complements the anti-tumor immune mechanisms induced by the therapeutic antibody. In this study, we explored different conjugation strategies for TLR9 agonists to immunoglobulin G (IgG). We evaluated biochemical conjugation of immunostimulatory CpG oligodesoxyribonucleotides (ODN) to the HER2-specific therapeutic antibody Trastuzumab with different cross-linkers comparing stochastic with site-specific conjugation. The physiochemical make-up and biological activities of the generated Trastuzumab-ODN conjugates were characterized in vitro and demonstrated that site-specific conjugation of CpG ODN is crucial for maintaining the antigen-binding capabilities of Trastuzumab. Furthermore, site-specific conjugate was effective in promoting anti-tumor immune responses in vivo in a pseudo-metastasis mouse model with engineered human HER2-transgenic tumor cells. In this in vivo model, co-delivery of Trastuzumab and CpG ODN in form of site-specific conjugates was superior to co-injection of unconjugated Trastuzumab, CpG ODN or stochastic conjugate in promoting T cell activation and expansion. Thereby, this study highlights that site-specific conjugation of CpG ODN to therapeutic antibodies targeting tumor markers is a feasible and more reliable approach for generation of conjugates which retain and combine the functional properties of the adjuvant and the antibody.

Plasmacytoid dendritic cell-derived type I interferon is crucial for the adjuvant activity of Toll-like receptor 7 agonists.

There is a high demand for the development of adjuvants that induce cytotoxic T lymphocytes, which are crucial for the elimination of intracellular pathogens and tumor cells. Toll-like receptor (TLR) agonists are prime candidates to fulfill this role because they induce innate immune activation and promote adaptive immune responses. The successful application of the TLR7 agonist R837 for treatment of basal cell carcinoma shows the potential for exploiting this pathway in tumor immunotherapy. Imidazoquinolines like R837 and stimulatory ssRNA oligonucleotides both trigger TLR7-mediated immune activation, but little is known about their comparative ability to promote immunity induction. We investigated differences in innate immune activation and adjuvant activity between the imidazoquinoline R848 and the ssRNA TLR7 agonist polyUs21. In contrast to R848, polyUs21 induced detectable levels of intracellular interferon-alpha (IFN-alpha) in plasmacytoid dendritic cells (PDCs). In immunization studies, only polyUs21 led to robust priming of type 1 T helper cells and cytotoxic T lymphocytes, and it was more efficient in inducing antitumor immunity than R848. Notably, exogenous IFN-alpha augmented the adjuvant activity of R848, whereas depletion of PDC abrogated the adjuvanticity of polyUs21. This study, therefore, identifies sufficient IFN-alpha production by PDC as an important determinant of vaccine efficacy.

Toll-like receptor 3 promotes cross-priming to virus-infected cells.

Cross-presentation of cell-associated antigens plays an important role in regulating CD8+ T cell responses to proteins that are not expressed by antigen-presenting cells (APCs). Dendritic cells are the principal cross-presenting APCs in vivo and much progress has been made in elucidating the pathways that allow dendritic cells to capture and process cellular material. However, little is known about the signals that determine whether such presentation ultimately results in a cytotoxic T cell (CTL) response (cross-priming) or in CD8+ T cell inactivation (cross-tolerance). Here we describe a mechanism that promotes cross-priming during viral infections. We show that murine CD8alpha+ dendritic cells are activated by double-stranded (ds)RNA present in virally infected cells but absent from uninfected cells. Dendritic cell activation requires phagocytosis of infected material, followed by signalling through the dsRNA receptor, toll-like receptor 3 (TLR3). Immunization with virus-infected cells or cells containing synthetic dsRNA leads to a striking increase in CTL cross-priming against cell-associated antigens, which is largely dependent on TLR3 expression by antigen-presenting cells. Thus, TLR3 may have evolved to permit cross-priming of CTLs against viruses that do not directly infect dendritic cells.

PolyI:C-induced reduction in uptake of soluble antigen is independent of dendritic cell activation.

Dendritic cells (DC) are key players in the initiation and modulation of adaptive immune responses due to their ability to acquire and present antigen and stimulate T cells. For the induction of effector T cell functions, antigen must be presented by activated DC. In this study, we have compared uptake of antigen by mouse DC in the presence of different Toll-like receptor (TLR) agonists, which are potent inducers of DC activation. Here we show that the reduction in uptake of soluble antigen in the presence of the viral double-stranded RNA (dsRNA) analogues polyinosinic-polycytidylic acid and Ampligen is independent of TLR-mediated DC activation. A reduction in antigen uptake by bone marrow-derived and splenic DC was also observed in response to other RNA homopolymers such as polyinosinic and polyguanylic acids, which are known inhibitors of scavenger receptor-mediated endocytosis. Pinocytosis and mannose receptor-mediated uptake of soluble antigen were not affected by any of the tested nucleic acids. The reduction in antigen uptake by dsRNA did not negatively influence the T cell stimulating properties of the DC. In summary, we conclude that the decrease in antigen endocytosis observed in the presence of a variety of TLR agonists is independent of TLR signalling and is caused by competition for specific surface receptors that are involved in the uptake of these TLR agonists and the antigen.

Viral infection switches non-plasmacytoid dendritic cells into high interferon producers.

Type I interferons (IFN-I) are important cytokines linking innate and adaptive immunity. Plasmacytoid dendritic cells make high levels of IFN-I in response to viral infection and are thought to be the major source of the cytokines in vivo. Here, we show that conventional non-plasmacytoid dendritic cells taken from mice infected with a dendritic-cell-tropic strain of lymphocytic choriomeningitis virus make similarly high levels of IFN-I on subsequent culture. Similarly, non-plasmacytoid dendritic cells secrete high levels of IFN-I in response to double-stranded RNA (dsRNA), a major viral signature, when the latter is introduced into the cytoplasm to mimic direct viral infection. This response is partially dependent on the cytosolic dsRNA-binding enzyme protein kinase R and does not require signalling through toll-like receptor (TLR) 3, a surface receptor for dsRNA. Furthermore, we show that sequestration of dsRNA by viral NS1 (refs 6, 7) explains the inability of conventional dendritic cells to produce IFN-I on infection with influenza. Our results suggest that multiple dendritic cell types, not just plasmacytoid cells, can act as specialized interferon-producing cells in certain viral infections, and reveal the existence of a TLR-independent pathway for dendritic cell activation that can be the target of viral interference.

Activation of dendritic cells by toll-like receptors and C-type lectins.

As sentinels of the immune system, dendritic cells (DC) scan their environment for the presence of pathogens. DC sense pathogens either directly or indirectly via endogenous factors such as cytokines and chemokines, which are produced by other cell types in response to infection. Although indirect signals in form of endogenous factors alert DC, direct activation of DC by pathogen-associated molecular patterns (PAMP) is crucial for the induction of primary T cell responses. Direct recognition of PAMP is mediated by pattern recognition receptors (PRR) such as Toll-like receptors (TLR) and C-type lectin receptors (CLR). The molecular patterns that are recognized by these receptors are indispensable for the life cycle of the pathogens, and their structure or cellular localization is different from that of the host. TLR detect cell-wall components of bacteria, fungi, and protozoa at the cell surface or bacterial and viral nucleic acid structures in a specialized endosomal compartment, while CLR that are involved in pattern recognition bind to carbohydrate structures associated with pathogens.

Application of carbon nanotubes in cancer vaccines: Achievements, challenges and chances.

Tumour-specific, immuno-based therapeutic interventions can be considered as safe and effective approaches for cancer therapy. Exploitation of nano-vaccinology to intensify the cancer vaccine potency may overcome the need for administration of high vaccine doses or additional adjuvants and therefore could be a more efficient approach. Carbon nanotube (CNT) can be described as carbon sheet(s) rolled up into a cylinder that is nanometers wide and nanometers to micrometers long. Stemming from the observed capacities of CNTs to enter various types of cells via diversified mechanisms utilising energy-dependent and/or passive routes of cell uptake, the use of CNTs for the delivery of therapeutic agents has drawn increasing interests over the last decade. Here we review the previous studies that demonstrated the possible benefits of these cylindrical nano-vectors as cancer vaccine delivery systems as well as the obstacles their clinical application is facing.

Recognition of viral single-stranded RNA by Toll-like receptors.

The Toll-like receptors (TLR), mediating innate immune activation upon recognition of viral nucleic acids, represent promising targets for the development of adjuvants. Therefore, there is great interest in unraveling the underlying mechanisms of ligand recognition. Studies aiming to identify which sequences, nucleic acid modifications and molecular moieties of viral nucleic acids trigger or inhibit TLR activation have allowed insights into this subject, yet there are still many aspects of innate recognition of viral nucleic acids which are only partially understood. This review discusses our current understanding of TLR-mediated recognition of viral single-stranded RNA (ssRNA) by TLR7 and TLR8. Oligoribonucleotides (ORN) and small immune response modifiers such as imidazoquinolines with agonist function have served as tools to study ligand recognition. In addition, there is increasing evidence that TLR-mediated recognition of mammalian ssRNA triggers innate immune activation and plays a role in autoimmunity. Thus the development of suitable TLR7 and TLR8 antagonists could pave the way for therapeutic intervention of particular autoimmune diseases.

Myosin II Synergizes with F-Actin to Promote DNGR-1-Dependent Cross-Presentation of Dead Cell-Associated Antigens.

Conventional type 1 DCs (cDC1s) excel at cross-presentation of dead cell-associated antigens partly because they express DNGR-1, a receptor that recognizes exposed actin filaments on dead cells. In vitro polymerized F-actin can be used as a synthetic ligand for DNGR-1. However, cellular F-actin is decorated with actin-binding proteins, which could affect DNGR-1 recognition. Here, we demonstrate that myosin II, an F-actin-associated motor protein, greatly potentiates the binding of DNGR-1 to F-actin. Latex beads coated with F-actin and myosin II are taken up by DNGR-1+ cDC1s, and antigen associated with those beads is efficiently cross-presented to CD8+ T cells. Myosin II-deficient necrotic cells are impaired in their ability to stimulate DNGR-1 or to serve as substrates for cDC1 cross-presentation to CD8+ T cells. These results provide insights into the nature of the DNGR-1 ligand and have implications for understanding immune responses to cell-associated antigens and for vaccine design.

Macrophages are exploited from an innate wound healing response to facilitate cancer metastasis.

Tumour-associated macrophages (TAMs) play an important role in tumour progression, which is facilitated by their ability to respond to environmental cues. Here we report, using murine models of breast cancer, that TAMs expressing fibroblast activation protein alpha (FAP) and haem oxygenase-1 (HO-1), which are also found in human breast cancer, represent a macrophage phenotype similar to that observed during the wound healing response. Importantly, the expression of a wound-like cytokine response within the tumour is clinically associated with poor prognosis in a variety of cancers. We show that co-expression of FAP and HO-1 in macrophages results from an innate early regenerative response driven by IL-6, which both directly regulates HO-1 expression and licenses FAP expression in a skin-like collagen-rich environment. We show that tumours can exploit this response to facilitate transendothelial migration and metastatic spread of the disease, which can be pharmacologically targeted using a clinically relevant HO-1 inhibitor.

Repurposing Tin Mesoporphyrin as an Immune Checkpoint Inhibitor Shows Therapeutic Efficacy in Preclinical Models of Cancer.

Purpose: Unprecedented clinical outcomes have been achieved in a variety of cancers by targeting immune checkpoint molecules. This preclinical study investigates heme oxygenase-1 (HO-1), an immunosuppressive enzyme that is expressed in a wide variety of cancers, as a potential immune checkpoint target in the context of a chemotherapy-elicited antitumor immune response. We evaluate repurposing tin mesoporphyrin (SnMP), which has demonstrated safety and efficacy targeting hepatic HO in the clinic for the treatment of hyperbilirubinemia, as an immune checkpoint blockade therapy for the treatment of cancer.Experimental Design: SnMP and genetic inactivation of myeloid HO-1 were evaluated alongside 5-fluorouracil in an aggressive spontaneous murine model of breast cancer (MMTV-PyMT). Single-cell RNA sequencing analysis, tumor microarray, and clinical survival data from breast cancer patients were used to support the clinical relevance of our observations.Results: We demonstrate that SnMP inhibits immune suppression of chemotherapy-elicited CD8+ T cells by targeting myeloid HO-1 activity in the tumor microenvironment. Microarray and survival data from breast cancer patients reveal that HO-1 is a poor prognostic factor in patients receiving chemotherapy. Single-cell RNA-sequencing analysis suggests that the myeloid lineage is a significant source of HO-1 expression, and is co-expressed with the immune checkpoints PD-L1/2 in human breast tumors. In vivo, we therapeutically compare the efficacy of targeting these two pathways alongside immune-stimulating chemotherapy, and demonstrate that the efficacy of SnMP compares favorably with PD-1 blockade in preclinical models.Conclusions: SnMP could represent a novel immune checkpoint therapy, which may improve the immunological response to chemotherapy. Clin Cancer Res; 24(7); 1617-28. ©2018 AACR.

Interleukin-10 and prostaglandin E2 have complementary but distinct suppressive effects on Toll-like receptor-mediated dendritic cell activation in ovarian carcinoma.

Dendritic cells (DC) have the potential to instigate a tumour-specific immune response, but their ability to prime naïve lymphocytes depends on their activation status. Thus, for tumour immunotherapy to be effective, the provision of appropriate DC activation stimuli such as Toll-like receptor (TLR) agonists is crucial in order to overcome immunosuppression associated with the tumour microenvironment. To address this, we investigated how ovarian carcinoma (OC)-associated ascites impedes activation of DC by TLR agonists. Our results show that ascites reduces the TLR-mediated up-regulation of CD86 and partially inhibits the production of the pro-inflammatory cytokines interleukin 6 (IL-6), IL-12 and tumour necrosis factor α (TNFα) in monocyte-derived DC from healthy controls. We further observe an impaired T cell stimulatory capacity of DC upon activation with TLR agonists in the presence of ascites, indicating that their functionality is affected by the immunosuppressive factors. We identify IL-10 and prostaglandin E2 (PGE2) as the pivotal immunosuppressive components in OC-associated ascites compromising TLR-mediated DC activation. Interestingly, IL-10 is present in both ascites from patients with malignant OC and in peritoneal fluid from patients with benign ovarian conditions and both fluids have similar ability to reduce TLR-mediated DC activation. However, depletion of IL-10 from ascites revealed that the presence of paracrine IL-10 is not crucial for ascites-mediated suppression of DC activation in response to TLR activation. Unlike IL-10, PGE2 is absent from peritoneal fluid of patients with benign conditions and selectively reduces TNFα induction in response to TLR-mediated activation in the presence of OC-associated ascites. Our study highlights PGE2 as an immunosuppressive component of the malignant OC microenvironment rendering PGE2 a potentially important target for immunotherapy in OC.

Dual stimulation of antigen presenting cells using carbon nanotube-based vaccine delivery system for cancer immunotherapy.

Although anti-cancer immuno-based combinatorial therapeutic approaches have shown promising results, efficient tumour eradication demands further intensification of anti-tumour immune response. With the emerging field of nanovaccinology, multi-walled carbon nanotubes (MWNTs) have manifested prominent potentials as tumour antigen nanocarriers. Nevertheless, the utilization of MWNTs in co-delivering antigen along with different types of immunoadjuvants to antigen presenting cells (APCs) has not been investigated yet. We hypothesized that harnessing MWNT for concurrent delivery of cytosine-phosphate-guanine oligodeoxynucleotide (CpG) and anti-CD40 Ig (αCD40), as immunoadjuvants, along with the model antigen ovalbumin (OVA) could potentiate immune response induced against OVA-expressing tumour cells. We initially investigated the effective method to co-deliver OVA and CpG using MWNT to the APC. Covalent conjugation of OVA and CpG prior to loading onto MWNTs markedly augmented the CpG-mediated adjuvanticity, as demonstrated by the significantly increased OVA-specific T cell responses in vitro and in C57BL/6 mice. αCD40 was then included as a second immunoadjuvant to further intensify the immune response. Immune response elicited in vitro and in vivo by OVA, CpG and αCD40 was significantly potentiated by their co-incorporation onto the MWNTs. Furthermore, MWNT remarkably improved the ability of co-loaded OVA, CpG and αCD40 in inhibiting the growth of OVA-expressing B16F10 melanoma cells in subcutaneous or lung pseudo-metastatic tumour models. Therefore, this study suggests that the utilization of MWNTs for the co-delivery of tumour-derived antigen, CpG and αCD40 could be a competent approach for efficient tumours eradication.

Carbon nanotubes' surface chemistry determines their potency as vaccine nanocarriers in vitro and in vivo.

Carbon nanotubes (CNTs) have shown marked capabilities in enhancing antigen delivery to antigen presenting cells. However, proper understanding of how altering the physical properties of CNTs may influence antigen uptake by antigen presenting cells, such as dendritic cells (DCs), has not been established yet. We hypothesized that altering the physical properties of multi-walled CNTs (MWNTs)-antigen conjugates, e.g. length and surface charge, can affect the internalization of MWNT-antigen by DCs, hence the induced immune response potency. For this purpose, pristine MWNTs (p-MWNTs) were exposed to various chemical reactions to modify their physical properties then conjugated to ovalbumin (OVA), a model antigen. The yielded MWNTs-OVA conjugates were long MWNT-OVA (~386nm), bearing net positive charge (5.8mV), or short MWNTs-OVA (~122nm) of increasing negative charges (-23.4, -35.8 or -39mV). Compared to the short MWNTs-OVA bearing high negative charges, short MWNT-OVA with the lowest negative charge demonstrated better cellular uptake and OVA-specific immune response both in vitro and in vivo. However, long positively-charged MWNT-OVA showed limited cellular uptake and OVA specific immune response in contrast to short MWNT-OVA displaying the least negative charge. We suggest that reduction in charge negativity of MWNT-antigen conjugate enhances cellular uptake and thus the elicited immune response intensity. Nevertheless, length of MWNT-antigen conjugate might also affect the cellular uptake and immune response potency; highlighting the importance of physical properties as a consideration in designing a MWNT-based vaccine delivery system.

Plant virus particles carrying tumour antigen activate TLR7 and Induce high levels of protective antibody.

Induction of potent antibody is the goal of many vaccines targeted against infections or cancer. Modern vaccine designs that use virus-like particles (VLP) have shown efficacy for prophylactic vaccination against virus-associated cancer in the clinic. Here we used plant viral particles (PVP), which are structurally analogous to VLP, coupled to a weak idiotypic (Id) tumour antigen, as a conjugate vaccine to induce antibody against a murine B-cell malignancy. The Id-PVP vaccine incorporates a natural adjuvant, the viral ssRNA, which acts via TLR7. It induced potent protective anti-Id antibody responses in an in vivo mouse model, superior to the "gold standard" Id vaccine, with prevalence of the IgG2a isotype. Combination with alum further increased antibody levels and maintained the IgG2a bias. Engagement of TLR7 in vivo was followed by secretion of IFN-α by plasmacytoid dendritic cells and by activation of splenic CD11chi conventional dendritic cells. The latter was apparent from up-regulation of co-stimulatory molecules and from secretion of a wide range of inflammatory cytokines and chemokines including the Th1-governing cytokine IL-12, in keeping with the IgG2a antibody isotype distribution. PVP conjugates are a novel cancer vaccine design, offering an attractive molecular form, similar to VLP, and providing T-cell help. In contrast to VLP, they also incorporate a safe "in-built" ssRNA adjuvant.

Nucleic acids and endosomal pattern recognition: how to tell friend from foe?

The innate immune system has evolved endosomal and cytoplasmic receptors for the detection of viral nucleic acids as sensors for virus infection. Some of these pattern recognition receptors (PRR) detect features of viral nucleic acids that are not found in the host such as long stretches of double-stranded RNA (dsRNA) and uncapped single-stranded RNA (ssRNA) in case of Toll-like receptor (TLR) 3 and RIG-I, respectively. In contrast, TLR7/8 and TLR9 are unable to distinguish between viral and self-nucleic acids on the grounds of distinct molecular patterns. The ability of these endosomal TLR to act as PRR for viral nucleic acids seems to rely solely on the mode of access to the endolysosomal compartment in which recognition takes place. The current dogma states that self-nucleic acids do not enter the TLR-sensing compartment under normal physiological conditions. However, it is still poorly understood how dendritic cells (DC) evade activation by self-nucleic acids, in particular with regard to specific DC subsets, which are specialized in taking up material from dying cells for cross-presentation of cell-associated antigens. In this review we discuss the current understanding of how the immune system distinguishes between foreign and self-nucleic acids and point out some of the key aspects that still require further research and clarification.

Antibody-antigen-adjuvant conjugates enable co-delivery of antigen and adjuvant to dendritic cells in cis but only have partial targeting specificity.

Antibody-antigen conjugates, which promote antigen-presentation by dendritic cells (DC) by means of targeted delivery of antigen to particular DC subsets, represent a powerful vaccination approach. To ensure immunity rather than tolerance induction the co-administration of a suitable adjuvant is paramount. However, co-administration of unlinked adjuvant cannot ensure that all cells targeted by the antibody conjugates are appropriately activated. Furthermore, antigen-presenting cells (APC) that do not present the desired antigen are equally strongly activated and could prime undesired responses against self-antigens. We, therefore, were interested in exploring targeted co-delivery of antigen and adjuvant in cis in form of antibody-antigen-adjuvant conjugates for the induction of anti-tumour immunity. In this study, we report on the assembly and characterization of conjugates consisting of DEC205-specific antibody, the model antigen ovalbumin (OVA) and CpG oligodeoxynucleotides (ODN). We show that such conjugates are more potent at inducing cytotoxic T lymphocyte (CTL) responses than control conjugates mixed with soluble CpG. However, our study also reveals that the nucleic acid moiety of such antibody-antigen-adjuvant conjugates alters their binding and uptake and allows delivery of the antigen and the adjuvant to cells partially independently of DEC205. Nevertheless, antibody-antigen-adjuvant conjugates are superior to antibody-free antigen-adjuvant conjugates in priming CTL responses and efficiently induce anti-tumour immunity in the murine B16 pseudo-metastasis model. A better understanding of the role of the antibody moiety is required to inform future conjugate vaccination strategies for efficient induction of anti-tumour responses.

Monitoring of in vivo function of superparamagnetic iron oxide labelled murine dendritic cells during anti-tumour vaccination.

Dendritic cells (DCs) generated in vitro to present tumour antigens have been injected in cancer patients to boost in vivo anti-tumour immune responses. This approach to cancer immunotherapy has had limited success. For anti-tumour therapy, delivery and subsequent migration of DCs to lymph nodes leading to effective stimulation of effector T cells is thought to be essential. The ability to non-invasively monitor the fate of adoptively transferred DCs in vivo using magnetic resonance imaging (MRI) is an important clinical tool to correlate their in vivo behavior with response to treatment. Previous reports of superparamagnetic iron oxides (SPIOs) labelling of different cell types, including DCs, have indicated varying detrimental effects on cell viability, migration, differentiation and immune function. Here we describe an optimised labelling procedure using a short incubation time and low concentration of clinically used SPIO Endorem to successfully track murine DC migration in vivo using MRI in a mouse tumour model. First, intracellular labelling of bone marrow derived DCs was monitored in vitro using electron microscopy and MRI relaxometry. Second, the in vitro characterisation of SPIO labelled DCs demonstrated that viability, phenotype and functions were comparable to unlabelled DCs. Third, ex vivo SPIO labelled DCs, when injected subcutaneously, allowed for the longitudinal monitoring by MR imaging of their migration in vivo. Fourth, the SPIO DCs induced the proliferation of adoptively transferred CD4(+) T cells but, most importantly, they primed cytotoxic CD8(+) T cell responses to protect against a B16-Ova tumour challenge. Finally, using anatomical information from the MR images, the immigration of DCs was confirmed by the increase in lymph node size post-DC injection. These results demonstrate that the SPIO labelling protocol developed in this study is not detrimental for DC function in vitro and in vivo has potential clinical application in monitoring therapeutic DCs in patients with cancer.

Determination of T-cell fate by dendritic cells.

Dendritic cells (DC) are professional antigen-presenting cells with a unique T-cell stimulatory aptitude that play a crucial role in the instruction of adaptive immune responses upon infection. By controlling the initiation of a diverse set of effector functions, which are suitable for the elimination of a wide range of pathogens, DCs form the pivotal link between the innate and the adaptive immune system. The innate pattern recognition pathways that trigger DC activation are central for skewing of the adaptive immune responses that are subsequently induced. Thus innate activation not only precedes adaptive immune activation, it also controls it and tailors the effector functions to the requirements of the infection. The adaptive immune response has to match the nature of the infection, but this does not only concern the type of pathogen, it is also affected by the localization of the infection. Tissue homeostasis has to be ensured and thus tissue-derived environmental factors influence the functional activity of activated DCs and thereby contribute to shaping of the immune response. Adaptive immune responses are vital for the elimination of pathogens, have the potential to attack tumor cells and play a detrimental role during transplant rejection and in a variety of autoimmune diseases. Better understanding of the mechanisms that control the induction of different T-cell effector functions will enable the development of strategies to manipulate the immune system in the context of vaccination, tumor immunotherapy, transplantation and autoimmunity.