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⁺ 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⁺ and CD8⁺ 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. ©2012 AACR.

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-based chemotherapeutics markedly reduced expression of the T cell inhibitory molecule programmed death receptor-ligand 2 (PD-L2) on both human DCs and human tumor cells. Downregulation of PD-L2 resulted in enhanced antigen-specific proliferation and Th1 cytokine secretion as well as enhanced recognition of tumor cells by T cells. Further analysis revealed that STAT6 controlled downregulation of PD-L2. Consistent with these data, patients with STAT6-expressing head and neck cancer displayed enhanced recurrence-free survival upon treatment with cisplatin-based chemoradiation compared with patients with STAT6-negative tumors, demonstrating the clinical relevance of platinum-induced STAT6 modulation. We therefore conclude that platinum-based anticancer drugs can enhance the immunostimulatory potential of DCs and decrease the immunosuppressive capability of tumor cells. This dual action of platinum compounds may extend their therapeutic application in cancer patients and provides a rationale for their use in combination with immunostimulatory compounds.

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 cytoskel‐eton 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.—Schreibelt, G., Kooij, G., Reijerkerk, A., van Doorn, R., Gringhuis, S. I., van der Pol, S., Weksler, B. B., Romero, I. A., Couraud, P.‐O., Piontek, J., Blasig, I. E., Dijkstra, C. D., Ronken, E., de Vries, H. E. Reactive oxygen species alter brain endothelial tight junction dynamics via RhoA, PI3 kinase and PKB signaling. FASEB J. 21, 3666–3676 (2007)

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.