Cross-presentation allows antigen-presenting cells to present exogenous antigens to CD8⁺ T cells, playing an essential role in controlling infections and tumor development. IFN-α induces the rapid differentiation of human mono-cytes into dendritic cells, known as IFN-DCs, highly efficient in mediating cross-presentation, as well as the cross-priming of CD8⁺ T cells. Here, we have investigated the mechanisms underlying the cross-presentation ability of IFN-DCs by studying the intracellular sorting of soluble ovalbumin and nonstructural-3 protein of hepatitis C virus. Our results demonstrate that, independently from the route and mechanism of antigen entry, IFN-DCs are extraordinarily competent in preserving internalized proteins from early degradation and in routing antigens toward the MHC class-I processing pathway, allowing long-lasting, cross-priming capacity. In IFN-DCs, both early and recycling endosomes function as key compartments for the storage of both antigens and MHC-class I molecules and for proteasome- and transporter-associated with Ag processing–dependent auxiliary cross-presentation pathways. Because IFN-DCs closely resemble human DCs naturally occurring in vivo in response to infections and other danger signals, these findings may have important implications for the design of vaccination strategies in neoplastic or chronic infectious diseases.

Dendritic cells (DC) generated after a short‐term exposure of monocytes to IFN‐α 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.

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.

The 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-cis-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.

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 IFNα 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 IFNγ 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.