Dendritic cells (DC) generated after a short-term exposure of monocytes to IFN-a and GM-CSF (IFN-DC) are highly effective in inducing cross-priming of CD8 + T cells against viral antigens. We have investigated the mechanisms responsible for the special attitude of these DC and compared their activity with that of reference DC. Antigen uptake and endosomal processing capabilities were similar for IFN-DC and IL-4-derived DC. Both DC types efficiently cross-presented soluble HCV NS3 protein to the specific CD8 + T cell clone, even though IFN-DC were superior in cross-presenting low amounts of viral antigens. Moreover, when DC were pulsed with inactivated HIV-1 and injected into hu-PBL-SCID mice, the generation of virus-specific CD8 + T cells was markedly higher in animals immunized with IFN-DC than in mice immunized with CD40L-matured IL-4-DC. Of interest, in experiments with purified CD8 + T cells, IFN-DC were superior with respect to CD40L-matured IL-4-DC in inducing in vitro cross-priming of HIV-specific CD8 + T cells. This property correlated with enhanced potential to express the specific subunits of the IL-23 and IL-27 cytokines. These results suggest that IFN-DC are directly licensed for an efficient CD8 + T cell priming by mechanisms likely involving enhanced antigen presentation and special attitude to produce IL-12 family cytokines.

IntroductionOver the past years, it has become apparent that type-I interferons (IFNs) affect adaptive immunity through their effects on monocytes. In particular, IFN-␣ has been shown to act as a potent inducer of the rapid differentiation of human monocytes into highly activated and partially mature dendritic cells (DCs), known as IFN-DCs. 1 We demonstrated previously that human monocytes exposed to granulocyte macrophage colony-stimulating factor (GM-CSF) and IFN-␣ are rapidly induced to express a set of membrane molecules involved in antigen (Ag) presentation and T-cell costimulation, as well as to strongly promote T helper (Th)-1 response and CD8 ϩ T cell cross-priming. 2 Moreover, IFN-DCs were shown to cross-present very efficiently low amounts of nonstructural-3 protein (NS3) of hepatitis C virus (HCV) to a specific CD8 ϩ T cell clone, even in the absence of CD4 ϩ T-cell help. 2 The cross-presentation efficiency of DCs is not dictated solely by their Ag capture capability 3 ; it also is affected by critical factors such as (1) the route of Ag uptake, (2) acidification-sensitive Ag degradation in endosomal-lysosomal compartments, and (3) Ag entry into the major histocompatibility complex class-I (MHC-I) pathway. [4][5][6][7][8] At present, 4 nonmutually exclusive models have been proposed to explain cross-presentation. [8][9][10] In the canonical cytosolic pathway, endocytosed Ags are translocated into the cytosol, where they are degraded by the proteasome, and then the antigenic peptides are transported into the lumen of the endoplasmic reticulum (ER) by the transporters associated with Ag processing (TAPs), 9,10 or alternatively, reimported from the cytoplasm into the early endosomes and loaded onto endosomal 12 According to a less well-defined TAP-and proteasomeindependent endosomal pathway, Ags can be processed by endosomal proteases and loaded onto MHC-I molecules directly within early and late endosomes and lysosomes. [13][14][15] An additional model involves the delivery of components of the ER to endocytic organelles or the transport of incoming Ags to the ER. [16][17][18] Here, we have investigated the mechanisms underlying the superior efficiency of IFN-DCs in the cross-presentation of soluble proteins, by studying (1) the Ag uptake and trafficking to the class-I processing pathway, (2) the maturation kinetic of the organelles containing the internalized proteins, (3) the Ag stability within endosomes, and (4) the Ag processing and cross-presentation to specific CD8 ϩ T cells. The results reveal that IFN-DCs exhibit a delayed endosomal acidification associated with a prolonged Ag survival and retention in the early endosomal compartment, as well as with Ag trafficking to recycling pathways. In IFN-DCs, both early and recycling endosomal compartments serve as important stores of MHC-I molecules, allowing rapid presentation of exogenous Ags. These findings provide novel mechanistic insight into the cross-presentation efficiency of IFN-DCs and underscore the potential advantage of using these cellula...

Gold nanoparticles (AuNPs) have been extensively used in biological applications because of their high\ud biocompatibility, ease of characterization and the extensive knowledge of their surface chemistry. These\ud features make AuNPs readily exploitable for drug delivery and novel diagnostic and therapeutic\ud approaches. In a previous work, we showed that small size (5–10 nm) AuNPs functionalized by sodium\ud 3-mercapto-1-propanesulfonate (3MPS) can be efficiently loaded with the glucocorticoid drug\ud dexamethasone (DXM) and are stable in water and PBS. In the present study, we further analysed the\ud stability and the drug release kinetics of DXM-loaded AuNPs functionalized by sodium 3mercaptopropane\ud sulfonate (AuNP-3MPS/DXM) and their unconjugated counterparts (AuNP-3MPS) in\ud different biological media. Moreover, we evaluated AuNP-3MPS cyto-compatibility on two mammalian\ud cell lines and tested their specific activity as drug carriers on DXM-sensitive murine and human tumor\ud cells. The colloidal stability of AuNP-3MPS/DXM was significantly increased in all tested culture media,\ud compared with the unconjugated AuNP-3MPS and both AuNP-3MPS formulations which proved nontoxic\ud to biological systems in vitro. Most importantly, we showed that AuNP-3MPS/DXM continuously\ud release bioactive DXM molecules that efficiently induce cell proliferation arrest and apoptotic cell death\ud on a human lymphoma cell line and upregulation of the DXM-inducible programmed cell death-1 (PD-1)\ud molecule on activated mouse T lymphocytes. These data confirm that the AuNP-3MPS/DXM conjugate\ud is a promising system for drug delivery and open interesting perspectives for future in vivo applications

SummaryThe capacity of the immunomodulatory drug rapamycin (RAPA) to inhibit replication of the CCR5 strain of human immunodeficiency virus (HIV) in vitro prompted us to test its effects in a murine preclinical model of HIV infection. RAPA (0·6 or 6 mg/kg body weight) or its vehicle were administered daily, per os, to SCID mice reconstituted with human peripheral blood leucocytes (hu-PBL) starting 2 days before the intraperitoneal challenge with the R5 tropic SF162 strain of HIV-1 (1000 50% tissue culture infective dose/ml). Relative to hu-PBL-SCID mice that received no treatment, HIV-infected hu-PBL-SCID mice treated with the vehicle control for 3 weeks exhibited a severe depletion of CD4 + cells (90%), an increase in CD8 + cells and an inversion of the CD4 + /CD8 + cell ratio. In contrast, treatment of HIV-infected mice with RAPA prevented a decrease in CD4 + cells and the increase of CD8 + cells, thereby preserving the original CD4+ : CD8 + cell ratio. Viral infection also resulted in the detection of HIV-DNA within peritoneal cells and spleen, and lymph node tissues of the vehicle-treated mice within 3 weeks of the viral challenge. In contrast, treatment with RAPA decreased cellular provirus integration and reduced HIV-RNA levels in the blood. Furthermore, in co-cultivation assays, spleens from RAPA-treated mice exhibited a reduced capacity for infecting allogeneic T cells which was dose-dependent. These data show that RAPA possesses powerful anti-viral activity against R5 strains of HIV in vivo and support the use of additional studies to evaluate the potential application of this drug in the management of HIV patients.

Although promising, the clinical benefit provided by dendritic cell (DC)-based vaccines is still limited and the choice of the optimal antigen formulation is still an unresolved issue. We have developed a new DC-based vaccination protocol for aggressive and/or refractory lymphomas which combines the unique features of interferon-conditioned DC (IFN-DC) with highly immunogenic tumor cell lysates (TCL) obtained from lymphoma cells undergoing immunogenic cell death. We show that treatment of mantle cell lymphoma (MCL) and diffuse large B-cell lymphoma (DLBCL) cell lines with 9--retinoic acid and IFNα (RA/IFNα) induces early membrane exposure of Calreticulin, HSP70 and 90 together with CD47 down-regulation and enhanced HMGB1 secretion. Consistently, RA/IFNα-treated apoptotic cells and -TCLs were more efficiently phagocytosed by DCs compared to controls. Notably, cytotoxic T cells (CTLs) generated with autologous DCs pulsed with RA/IFNα-TCLs more efficiently recognized and specifically lysed MCL or DLBCL cells or targets loaded with several HLA-A*0201 cyclin D1 or HLA-B*0801 survivin epitopes. These cultures also showed an expansion of Th1 and Th17 cells and an increased Th17/Treg ratio. Moreover, DCs loaded with RA/IFNα-TCLs showed enhanced functional maturation and activation. NOD/SCID mice reconstituted with human peripheral blood lymphocytes and vaccinated with autologous RA/IFNα-TCL loaded-IFN-DCs showed lymphoma-specific T-cell responses and a significant decrease in tumor growth with respect to mice treated with IFN-DC unpulsed or loaded with untreated TCLs. This study demonstrates the feasibility and efficacy of the use of RA/IFNα to generate a highly immunogenic TCL as a suitable tumor antigen formulation for the development of effective anticancer DC-based vaccines.

IFN-α exerts multiple effects leading to immune protection against pathogens and cancer as well to autoimmune reactions by acting on monocytes and dendritic cells. We analyzed the versatility of human monocytes conditioned by IFN-α towards dendritic cell differentiation (IFN-DC) in shaping the autologous T-helper response. Priming of naïve CD4 T cells with autologous IFN-DC in the presence of either SEA or anti-CD3, resulted, in addition to a prominent expansion of CXCR3+ IFN-γ-producing CD4 Th1 cells, in the emergence of two distinct subsets of IL-17-producing CD4 T cells: i) a predominant Th17 population selectively producing IL-17 and expressing CCR6; ii) a minor Th1/Th17 population, producing both IL-17 and IFN-γ. After phagocytosis of apoptotic cells, IFN-DC induced Th17 cell expansion and IL-17 release. Notably, the use of neutralizing antibodies revealed that IL-23 was an essential cytokine in mediating Th17 cell development by IFN-DC. The demonstration of the IFN-DC-induced expansion of both Th1 and Th17 cell populations reveals the intrinsic plasticity of these DC in orienting the immune response and provides a mechanistic link between IFN-α and the onset of autoimmune phenomena, which have been correlated with both IL-17 production and exposure to IFN-α.

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Purpose: This study was aimed at evaluating the feasibility, safety, immunologic and clinical responses in patients with follicular lymphoma treated with monocyte-derived dendritic cells generated in the presence of IFNa and GM-CSF (IFN-DC) in combination with low doses of rituximab.Patients and Methods: Firstly, we analyzed in vitro and in vivo the immunologic properties of IFN-DC against follicular lymphoma. Thus, we performed a phase I trial in 8 patients with refractory and relapsed follicular lymphoma based on sequential intranodal injections of low-dose of rituximab and unloaded IFN-DC and report the safety, clinical, and immunologic results of the enrolled patients.Results: Preclinical studies indicated that IFN-DC can synergize with rituximab leading to increased cytotoxicity and T-cell tumor infiltration. The clinical evaluation showed that the combined treatment was totally safe. The overall response rate was 50%, PET-negative complete response rate 37%, and remission is still ongoing in 2/4 of responding patients (median follow-up 26 months, range 11-47). Notably, following the combined therapy all patients showed induction/enhancement of T-cell responses by CD107 degranulation or IFNg ELISPOT assay against patient-specific tumor IGHV sequences.Conclusions: These results represent the proof-of-principle on the effectiveness of unloaded IFN-DC in inducing durable clinical responses and promoting induction of tumor-specific peripheral T cells, thus suggesting the occurrence of an effective endogenous antitumor vaccination. The overall findings indicate that some unique properties of IFN-DC can be successfully exploited to induce/enhance antitumor responses, thus representing a valuable antitumor strategy for novel and more effective combination therapies in patients with cancer.