Type I interferons (IFNs) are cytokines exhibiting antiviral and antitumor effects, including multiple activities on immune cells. However, the importance of these cytokines in the early events leading to the generation of an immune response is still unclear. Here, we have investigated the effects of type I IFNs on freshly isolated granulocyte/macrophage colony-stimulating factor (GM-CSF)–treated human monocytes in terms of dendritic cell (DC) differentiation and activity in vitro and in severe combined immunodeficiency mice reconstituted with human peripheral blood leukocytes (hu-PBL-SCID) mice. Type I IFNs induced a surprisingly rapid maturation of monocytes into short-lived tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)–expressing DCs endowed with potent functional activities, superior with respect to the interleukin (IL)-4/GM-CSF treatment, as shown by FACS® analyses, mixed leukocyte reaction assays with allogeneic PBLs, and lymphocyte proliferation responses to HIV-1–pulsed autologous DCs. Type I IFN induced IL-15 production and strongly promoted a T helper cell type 1 response. Notably, injection of IFN-treated HIV-1–pulsed DCs in SCID mice reconstituted with autologous PBLs resulted in the generation of a potent primary immune response, as evaluated by the detection of human antibodies to various HIV-1 antigens. These results provide a rationale for using type I IFNs as vaccine adjuvants and support the concept that a natural alliance between these cytokines and monocytes/DCs represents an important early mechanism for connecting innate and adaptive immunity.

IntroductionDendritic cells (DCs) are the most potent antigen-presenting cells playing a pivotal role in the induction of the immune response. [1][2][3] DCs are located in peripheral tissues, in sites where they can optimally survey for incoming pathogens. The interaction of DCs with pathogens leads to migration to secondary lymphoid organs where they initiate a specific immune response. Notably, the migration capability of DCs is strictly regulated by their response to soluble factors, namely chemokines 4,5 that characterize maturation stage and shape functional activities of DCs.Chemokines represent a family of 8-to 10-kDa secreted proteins capable of regulating migration and activation not only of leukocytes, including DCs, but also of stromal cells. 6,7 It is well documented that migration of DCs is tightly regulated as a function of maturation. [8][9][10][11][12] In particular, immature DCs respond to many CC and CXC chemokines, such as MIP-1␣, MIP-1␤, RANTES, and MIP-3␣, whereas mature DCs have lost their responsiveness to most of these chemokines, as a result of down-regulation of receptor expression or activity. However, mature DCs have been reported to respond to MIP-3␤/ELC and 6Ckine/SLC as a consequence of an up-regulation of their receptor (CCR7). Of interest, studies in knock-out mice for CCR7 have shown the crucial importance of the CCR7/MIP-3␤ interaction for the generation of a primary immune response. 13 All this emphasizes the essential role of certain chemokines/chemokine receptors and DC migration properties in the generation of the immune response.DCs are derived from hematopoietic progenitor cells, 2,3 and distinct subtypes of human circulating DCs have been detected in the blood. 14,15 However, the mechanisms regulating generation, functions, and survival of blood-circulating DCs in response to infections are largely unknown. The rapid generation of active DCs endowed with potent migratory capabilities would be advantageous for a prompt immune response to incoming pathogens.Blood monocytes are highly versatile cells playing crucial roles in the maintenance of immune homeostasis. These cells circulate in the bloodstream, transmigrate through vascular endothelium, and localize in peripheral and mucosal tissues, where they differentiate into different cell types. 16,17 Monocyte-derived mature DCs are currently generated in vitro by 2 sequential treatments, 18 leading first to the so-called "immature DCs," after exposure for several days to both granulocytemacrophage colony-stimulating factor (GM-CSF) and interleukin 4 (IL-4), and then to mature DCs, after a subsequent addition of stimuli such as lipopolysaccharide (LPS), CD40L, or virus infection. However, the in vivo relevance of monocyte differentiation into DCs remains unclear, especially because exposure of monocytes to IL-4 can hardly mimic the cytokine milieu likely to be present under in vivo conditions at the infection site.Although results indicate that DC maturation can occur directly from monocytes during transendothelial migration...

contributed equally to this work CD95 (APO-1/Fas) is a member of the tumor necrosis factor receptor family, which can trigger apoptosis in a variety of cell types. However, little is known of the mechanisms underlying cell susceptibility to CD95-mediated apoptosis. Here we show that human T cells that are susceptible to CD95-mediated apoptosis, exhibit a constitutive polarized morphology, and that CD95 colocalizes with ezrin at the site of cellular polarization. In fact, CD95 co-immunoprecipitates with ezrin exclusively in lymphoblastoid CD4 + T cells and primary long-term activated T lymphocytes, which are prone to CD95-mediated apoptosis, but not in short-term activated T lymphocytes, which are refractory to the same stimuli, even expressing equal levels of CD95 on the cell membrane. Pre-treatment with ezrin antisense oligonucleotides speci®cally protected from the CD95-mediated apoptosis. Moreover, we show that the actin cytoskeleton integrity is essential for this function. These ®ndings strongly suggest that the CD95 cell membrane polarization, through an ezrin-mediated association with the actin cytoskeleton, is a key intracellular mechanism in rendering human T lymphocytes susceptible to the CD95-mediated apoptosis. Keywords: apoptosis/CD95 Fas/cytoskeleton/ezrin/ polarization IntroductionBetween the major programmed cell death (PCD) pathways, CD95 (APO-1/Fas)-mediated apoptosis seems one of the most involved in both the physiological control of cell proliferation and in the pathogenesis of viral, autoimmune and neoplastic diseases (Linch et al., 1995;Giordano et al., 1997; Apoptosis, special section, 1998;Peter and Krammer, 1998). Particularly, the CD95 interaction with its ligand (FasL) plays a crucial role in homeostasis and self-tolerance of lymphocytes in both humans and mice (Nagata and Suda, 1995). However, despite abundant surface expression, cells may be either susceptible or refractory to CD95-mediated PCD (Klas et al., 1993;Suda et al., 1997). In fact, susceptibility to the CD95-mediated apoptosis may not be merely due to the surface expression of the CD95 antigen, in that lymphocytes equally expressing CD95 on the membrane are differently triggerable to PCD (Klas et al., 1993;Alderson et al., 1995;Brunner et al., 1995). Intracellular mechanisms involved in the positive or negative regulation of the CD95-signaling pathway have been described (Tschopp et al., 1998). However, cellular susceptibility to CD95-mediated PCD remains an unresolved issue and the search for novel mechanisms is necessary. Asymmetric organization of the plasma membrane and cytosolic organelles is fundamental for a variety of cells, including pro-and eukaryotic cells (Nelson, 1992). The degree to which cells polarize is characterized by their ability to create and maintain morphologically and biochemically distinct plasma membrane domains. The prototype of stable polarized membrane domains are the apical and basolateral surfaces of simple epithelial cells. However, T lymphocytes continuously change their shape and polariza...

A major challenge of AIDS research is the development of therapeutic vaccine strategies capable of inducing the humoral and cellular arms of the immune responses against HIV-1. In this work, we evaluated the capability of DCs pulsed with aldrithiol-2–inactivated HIV-1 in inducing a protective antiviral human immune response in SCID mice reconstituted with human PBL (hu-PBL-SCID mice). Immunization of hu-PBL-SCID mice with DCs generated after exposure of monocytes to GM-CSF/IFN-α (IFN-DCs) and pulsed with inactivated HIV-1 resulted in a marked induction of human anti–HIV-1 antibodies, which was associated with the detection of anti-HIV neutralizing activity in the serum. This vaccination schedule also promoted the generation of a human CD8+ T cell response against HIV-1, as measured by IFN-γ Elispot analysis. Notably, when the hu-PBL-SCID mice immunized with antigen-pulsed IFN-DCs were infected with HIV-1, inhibition of virus infection was observed as compared with control animals. These results suggest that IFN-DCs pulsed with inactivated HIV-1 can represent a valuable approach of immune intervention in HIV-1–infected patients.

Immunotherapy efficacy relies on the crosstalk within the tumor microenvironment between cancer and dendritic cells (DCs) resulting in the induction of a potent and effective antitumor response. DCs have the specific role of recognizing cancer cells, taking up tumor antigens (Ags) and then migrating to lymph nodes for Ag (cross)-presentation to naïve T cells. Interferon-α-conditioned DCs (IFN-DCs) exhibit marked phagocytic activity and the special ability of inducing Ag-specific T-cell response. Here, we have developed a novel microfluidic platform recreating tightly interconnected cancer and immune systems with specific 3D environmental properties, for tracking human DC behaviour toward tumor cells. By combining our microfluidic platform with advanced microscopy and a revised cell tracking analysis algorithm, it was possible to evaluate the guided efficient motion of IFN-DCs toward drug-treated cancer cells and the succeeding phagocytosis events. Overall, this platform allowed the dissection of IFN-DC-cancer cell interactions within 3D tumor spaces, with the discovery of major underlying factors such as CXCR4 involvement and underscored its potential as an innovative tool to assess the efficacy of immunotherapeutic approaches.

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Dendritic cells (DCs) are the most potent antigen-presenting cells (APCs) and thus play a pivotal role in induction of the immune response. Recent studies in both human and mouse models have shown that type I IFN, cytokines originally characterized for their antiviral activity and exerting multiple biologic effects, efficiently promote the differentiation and activation of DCs. These observations, together with the findings that DCs can express biologically relevant levels of type I interferon (IFN) and, in particular, that high amounts of these cytokines are released by specialized DC precursors (i.e., plasmacytoid DCs) in response to viral infections, strongly suggest the existence of a natural alliance between type I IFN and DCs, which is instrumental in ensuring an efficient immune response to both infectious agents and tumors. Further recent knowledge on the interactions between type I IFN and DCs emphasizes the importance of these cytokines in linking innate and adaptive immunity and may lead to new perspectives in their use as vaccine adjuvants as well as in strategies for the development of DC-based vaccines.

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