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.

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.

Enhanced cerebrovascular permeability and cellular infiltration mark the onset of early multiple sclerosis lesions. So far, the precise sequence of these events and their role in lesion formation and disease progression remain unknown. Here we provide quantitative evidence that blood-brain barrier leakage is an early event and precedes massive cellular infiltration in the development of acute experimental allergic encephalomyelitis (EAE), the animal correlate of multiple sclerosis. Cerebrovascular leakage and monocytes infiltrates were separately monitored by quantitative in vivo MRI during the course of the disease. Magnetic resonance enhancement of the contrast agent gadolinium diethylenetriaminepentaacetate (Gd-DTPA), reflecting vascular leakage, occurred concomitantly with the onset of neurological signs and was already at a maximal level at this stage of the disease. Immunohistochemical analysis also confirmed the presence of the serum-derived proteins such as fibrinogen around the brain vessels early in the disease, whereas no cellular infiltrates could be detected. MRI further demonstrated that Gd-DTPA leakage clearly preceded monocyte infiltration as imaged by the contrast agent based on ultra small particles of iron oxide (USPIO), which was maximal only during full-blown EAE. Ultrastructural and immunohistochemical investigation revealed that USPIOs were present in newly infiltrated macrophages within the inflammatory lesions. To validate the use of USPIOs as a non-invasive tool to evaluate therapeutic strategies, EAE animals were treated with the immunomodulator 3-hydroxy-3-methylglutaryl Coenzyme A reductase inhibitor, lovastatin, which ameliorated clinical scores. MRI showed that the USPIO load in the brain was significantly diminished in lovastatin-treated animals. Data indicate that cerebrovascular leakage and monocytic trafficking into the brain are two distinct processes in the development of inflammatory lesions during multiple sclerosis, which can be monitored on-line with MRI using USPIOs and Gd-DTPA as contrast agents. These studies also implicate that USPIOs are a valuable tool to visualize monocyte infiltration in vivo and quantitatively assess the efficacy of new therapeutics like lovastatin.

The blood-brain barrier (BBB) prevents the entrance of circulating molecules and immune cells into the central nervous system. The barrier is formed by specialized brain endothelial cells that are interconnected by tight junctions (TJ). A defective function of the BBB has been described for a variety of neuroinflammatory diseases, indicating that proper regulation is essential for maintaining brain homeostasis. Under pathological conditions, reactive oxygen species (ROS) significantly contribute to BBB dysfunction and inflammation in the brain by enhancing cellular migration. However, a detailed study about the molecular mechanism by which ROS alter BBB integrity has been lacking. Here we demonstrate that ROS alter BBB integrity, which is paralleled by cytoskeleton rearrangements and redistribution and disappearance of TJ proteins claudin-5 and occludin. Specific signaling pathways, including RhoA and PI3 kinase, mediated observed processes and specific inhibitors of these pathways prevented ROS-induced monocyte migration across an in vitro model of the BBB. Interestingly, these processes were also mediated by protein kinase B (PKB/Akt), a previously unknown player in cytoskeleton and TJ dynamics that acted downstream of RhoA and PI3 kinase. Our study reveals new insights into molecular mechanisms underlying BBB regulation and provides novel opportunities for the treatment of neuroinflammatory diseases.

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-

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Reactive oxygen species (ROS) contain one or more unpaired electrons and are formed as intermediates in a variety of normal biochemical reactions. However, when generated in excess amounts or not appropriately controlled, ROS initiate extensive cellular damage and tissue injury. ROS have been implicated in the progression of cancer, cardiovascular disease and neurodegenerative and neuroinflammatory disorders, such as multiple sclerosis (MS). In the last decade there has been a major interest in the involvement of ROS in MS pathogenesis and evidence is emerging that free radicals play a key role in various processes underlying MS pathology. To counteract ROS-mediated damage, the central nervous system is equipped with an intrinsic defense mechanism consisting of endogenous antioxidant enzymes. Here, we provide a comprehensive overview on the (sub)cellular origin of ROS during neuroinflammation as well as the detrimental effects of ROS in processing underlying MS lesion development and persistence. In addition, we will discuss clinical and experimental studies highlighting the therapeutic potential of antioxidant protection in the pathogenesis of MS.

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...