Vaccination against cancer by using dendritic cells has for more than a decade been based on dendritic cells generated ex vivo from monocytes or CD34 þ progenitors. Here, we report on the first clinical study of therapeutic vaccination against cancer using naturally occurring plasmacytoid dendritic cells (pDC). Fifteen patients with metastatic melanoma received intranodal injections of pDCs activated and loaded with tumor antigen-associated peptides ex vivo. In vivo imaging showed that administered pDCs migrated and distributed over multiple lymph nodes. Several patients mounted antivaccine CD4 þ and CD8 þ T-cell responses. Despite the limited number of administered pDCs, an IFN signature was observed after each vaccination. These results indicate that vaccination with naturally occurring pDC is feasible with minimal toxicity and that in patients with metastatic melanoma, it induces favorable immune responses. Cancer Res; 73(3); 1063-75. Ó2012 AACR.

The platinum-based drugs cisplatin, carboplatin, and oxaliplatin belong to the most widely used chemotherapeutics in oncology, showing clinical efficacy against many solid tumors. Their main mechanism of action is believed to be the induction of cancer cell apoptosis as a response to their covalent binding to DNA. In recent years, this picture has increased in complexity, based on studies indicating that cellular molecules other than DNA may potentially act as targets, and that part of the antitumor effects of platinum drugs occurs through modulation of the immune system. These immunogenic effects include modulation of STAT signaling; induction of an immunogenic type of cancer cell death through exposure of calreticulin and release of ATP and high-mobility group protein box-1 (HMGB-1); and enhancement of the effector immune response through modulation of programmed death receptor 1-ligand and mannose-6-phosphate receptor expression. Both basic and clinical studies indicate that at least part of the antitumor efficacy of platinum chemotherapeutics may be due to immune potentiating mechanisms. Clinical studies exploiting this novel mechanism of action of these old cancer drugs have been initiated. Here, we review the literature on the immunogenic effects of platinum, summarize the clinical advances using platinum as a cytotoxic compound with immune adjuvant properties, and discuss the limitations to these studies and the gaps in our understanding of the immunologic effects of these drugs.

Cellular therapy, including stem cell transplants and dendritic cell vaccines, is typically monitored for dosage optimization, accurate delivery and localization using non-invasive imaging, of which magnetic resonance imaging (MRI) is a key modality. 19 F MRI retains the advantages of MRI as an imaging modality, while allowing direct detection of labelled cells for unambiguous identification and quantification, unlike typical metal-based contrast agents. Recent developments in 19 F MRIbased in vivo cell quantification, the existing clinical use of 19 F compounds and current explosive interest in cellular therapeutics have brought 19 F imaging technology closer to clinical application.

Dendritic cell (DC) vaccination in cancer patients aims to induce or augment an effective antitumor immune response against tumor antigens and was first explored in a clinical trial in the 1990s. More than two decades later, numerous clinical trials have been performed or are ongoing with a wide variety of DC subsets, culture protocols, and treatment regimens. The safety of DC vaccination and its ability to induce antitumor responses have clearly been established; however, although scattered patients with long-term benefit were reported, DC vaccines have not yet fulfilled their promise, perhaps mainly due to the lack of large-scale well-conducted phase II/III trials. To allow meaningful multicenter phase III trials, the production of DC vaccines should be standardized between centers which is now becoming feasible. To improve the efficacy of DC-based immunotherapy, it could be combined with other treatments.

Tumor microenvironments feature immune inhibitory mechanisms that prevent T cells from generating effective antitumor immune responses. Therapeutic interventions aimed at disrupting these inhibitory mechanisms have been shown to enhance antitumor immunity, but they lack direct cytotoxic effects. Here, we investigated the effect of cytotoxic cancer chemotherapeutics on immune inhibitory pathways. We observed that exposure to platinum-

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. (Blood. 2012; 119(10):2284-2292) IntroductionDendritic cells (DCs) are central players in the induction of adaptive immune responses. 1 They reside in peripheral tissues, where they are positioned to capture antigens. In the immature state, DCs continuously sample their environment by receptormediated endocytosis, pinocytosis, and phagocytosis. Once DCs also encounter danger signals, such as those present in pathogens, endogenous danger molecules, inflammatory cytokines, or immune complexes, they become activated and migrate to the lymph nodes and differentiate into mature DCs, which is accompanied by stabilization of peptide-MHC complexes on the cell surface, up-regulation of costimulatory molecules, and cytokine release. These alterations contribute to optimal antigen presentation to T lymphocytes. DCs have the unique capacity to process extracellular antigens for cross-presentation via MHC class I. This feature allows DCs to induce CD8 T-cell responses against dying cells, tumor cells, and viruses that do not replicate in DCs.In human peripheral blood, 2 main populations of DCs can be distinguished: CD11c-positive myeloid DCs (mDCs) and CD11c-negative plasmacytoid DCs (pDCs). 2,3 Human mDCs can be subdivided further on the basis of differential surface expression of BDCA1 (CD1c), CD16, and BDCA3 (CD141). 4,5 Because the frequency of circulating mDCs in human blood is very low (Ͻ 2% of the peripheral blood leukocytes), many studies exploit in vitro-generated monocyte-derived DCs (moDCs) as "surrogate mDCs" because of the relative ease of obtaining large quantities of these cells. DC subsets are heterogeneous in the expression of cell surface markers and pathogen-recognition receptors, cytokine production after stimulation, as w...

Purpose: Thus far, dendritic cell (DC)-based immunotherapy of cancer was primarily based on in vitro-generated monocytederived DCs, which require extensive in vitro manipulation. Here, we report on a clinical study exploiting primary CD1c þ myeloid DCs, naturally circulating in the blood. Experimental Design: Fourteen stage IV melanoma patients, without previous systemic treatment for metastatic disease, received autologous CD1c þ myeloid DCs, activated by only brief (16 hours) ex vivo culture and loaded with tumor-associated antigens of tyrosinase and gp100. Results: Our results show that therapeutic vaccination against melanoma with small amounts (3-10 Â 10 6 ) of myeloid DCs is feasible and without substantial toxicity. Four of 14 patients showed long-term progression-free survival (12-35 months), which directly correlated with the development of multifunctional CD8þ T-cell responses in three of these patients. In particular, high CD107a expression, indicative for cytolytic activity, and IFNg as well as TNFa and CCL4 production was observed. Apparently, these T-cell responses are essential to induce tumor regression and promote long-term survival by stalling tumor growth. Conclusions:We show that vaccination of metastatic melanoma patients with primary myeloid DCs is feasible and safe and results in induction of effective antitumor immune responses that coincide with improved progression-free survival. Clin Cancer Res; 22(9); 2155-66. Ó2015 AACR.

Key Points• Cross-presentation of both soluble and cell-associated tumor antigens by human DC subsets is enhanced by addition of adjuvant TLR agonists. • Ability to cross-present exogenous antigen with high IFN␣ secretion puts human pDCs as activators of CD8 ϩ T cells in antitumor responses. IntroductionDendritic cells (DCs) are the professional antigen presenting cells (APCs) of the immune system with the unique capacity to attract and activate naive CD4 ϩ and CD8 ϩ T cells. 1 After infection or inflammation, DCs undergo a complex maturation process and migrate into lymph nodes where they present antigens (Ags) to T cells. The DC family is very heterogeneous and consists of different DC subsets, each with distinct functional characteristics. In human peripheral blood, at least 2 main populations of DCs can be distinguished: CD11c positive myeloid DCs (mDCs) and CD11c negative plasmacytoid DCs (pDCs). Myeloid DCs can be further subdivided based on the expression of CD16, CD1c, and BDCA3. 2 Transcriptional profiling revealed significant differences between the human blood DC subsets, 3 probably reflecting differences in their Ag-presenting capacities. Furthermore, mDCs and pDCs show clearly different responses to products derived from pathogens, as a result of their distinct Toll-like receptor (TLR) expression profiles. 4 Myeloid DCs have the capacity to produce IL-12 in response to microbial stimuli through TLRs, and thereby, induce Th1 responses. 5,6 Plasmacytoid DCs (pDCs), in contrast, are the key effectors in innate immunity because of their capacity to produce large amounts of type I IFNs in response to bacterial or viral infections. 7 Similar to mDC-derived IL-12, pDC-derived type I IFNs also participate in T-cell priming as Th1-inducing cytokines. 8 In addition to directing CD4 ϩ Th responses, DCs are also important for the generation of CD8 ϩ cytotoxic T-cell responses against viruses and tumors. As professional APCs, DCs have the unique capacity to take up, process, and present exogenously encountered Ags for cross-presentation via MHC class I molecules to CD8 ϩ T cells. Numerous studies have been performed to comprehend this cross-presentation process, and these have revealed 2 major pathways: (1) the "canonical" proteasome dependent cytosolic pathway, and (2) the TAP and proteasome independent pathway. [9][10][11][12] Many studies however, made use of murine DCs to study cross-presentation capacities and mechanisms used by different DC subsets. There is ample evidence that identified the CD8␣ ϩ DC as the superior cross-presenting DC subset in mice. 13,14 Recently, a lot of effort has been put toward finding the human counterpart of the murine cross-presenting CD8␣ ϩ DC subset. Despite basic similarities between human and mouse DCs, direct comparison is difficult because of large differences in cell-surface markers and TLR expression, in particular also for pDCs, which in contrast to mice are the sole TLR9-expressing subtype of DCs in Submitted June 7, 2012; accepted November 9, 2012. Prepublished onl...