Exploiting a new strategy to induce immunogenic cell death to improve dendritic cell-based vaccines for lymphoma immunotherapy.

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.

Immune-based mechanisms of cytotoxic chemotherapy: implications for the design of novel and rationale-based combined treatments against cancer.

Conventional anticancer chemotherapy has been historically thought to act through direct killing of tumor cells. This concept stems from the fact that cytotoxic drugs interfere with DNA synthesis and replication. Accumulating evidence, however, indicates that the antitumor activities of chemotherapy also rely on several off-target effects, especially directed to the host immune system, that cooperate for successful tumor eradication. Chemotherapeutic agents stimulate both the innate and adaptive arms of the immune system through several modalities: (i) by promoting specific rearrangements on dying tumor cells, which render them visible to the immune system; (ii) by influencing the homeostasis of the hematopoietic compartment through transient lymphodepletion followed by rebound replenishment of immune cell pools; (iii) by subverting tumor-induced immunosuppressive mechanisms and (iv) by exerting direct or indirect stimulatory effects on immune effectors. Among the indirect ways of immune cell stimulation, some cytotoxic drugs have been shown to induce an immunogenic type of cell death in tumor cells, resulting in the emission of specific signals that trigger phagocytosis of cell debris and promote the maturation of dendritic cells, ultimately resulting in the induction of potent antitumor responses. Here, we provide an extensive overview of the multiple immune-based mechanisms exploited by the most commonly employed cytotoxic drugs, with the final aim of identifying prerequisites for optimal combination with immunotherapy strategies for the development of more effective treatments against cancer.

Impaired myelopoiesis in mice devoid of interferon regulatory factor 1.

Interferon regulatory factor (IRF)-1 is a transcription factor controlling the expression of several genes, which are differentially induced depending on the cell type and signal. IRF-1 modulates multiple functions, including regulation of immune responses and host defence, cell growth, cytokine signalling and hematopoietic development. Here, we investigated the role of IRF-1 in granulocytic differentiation in mice with a null mutation in the IRF-1 gene. We show that IRF-1(-/-) bone marrow cells exhibit an increased number of immature granulocytic precursors, associated with a decreased number of mature granulocytic elements as compared to normal mice, suggestive of a defective maturation process. Clonogenetic analyses revealed a reduced number of CFU-G, CFU-M and CFU-GM colonies in IRF-1(-/-) mice, while the number of BFU-E/CFU-E colonies was unchanged. At the molecular level, the expression of CAAT-enhancer-binding protein (C/EBP)-epsilon, -alpha and PU.1 was substantially lower in the CD11b(+) cells from the bone marrow of IRF-1(-/-) mice as compared to cells from wild-type mice. These results, together with the fact that IRF-1 is markedly induced early during granulo-monocytic differentiation of CD34+ cells, highlight the pivotal role of IRF-1 in the early phases of myelopoiesis.

A new type I IFN-mediated pathway for the rapid differentiation of monocytes into highly active dendritic cells.

Dendritic cells (DCs) are a unique leukocyte type consisting of different subsets of professional antigen-presenting cells. Since DCs initiate and govern the immune response, they represent an ideal target for intervention aimed at modulating and potentiating immune responses against cancer and infectious diseases. We recently described and characterized, at a functional level, a novel DC subset, interferon (IFN)-DCs, derived from blood monocytes after a short exposure to type I IFN and GM-CSF. Here, we review our recent studies on IFN-DCs and discuss their possible use in clinical immunotherapeutic strategies.

Expression of CCR-7, MIP-3beta, and Th-1 chemokines in type I IFN-induced monocyte-derived dendritic cells: importance for the rapid acquisition of potent migratory and functional activities.

The migration capability of dendritic cells (DCs) is regulated by their response to factors, namely chemokines, that characterize maturation stage and shape their functional activities. This study examines the morphology, expression of chemokines/chemokine receptors, and migration properties of DCs generated after treatment of monocytes with type I interferon (IFN) and granulocyte-macrophage colony-stimulating factor (GM-CSF) (IFN-DCs). IFN-DCs showed phenotypical and morphologic features undetectable in DCs generated in the presence of interleukin 4 (IL-4) and GM-CSF, such as expression of CD83 and CD25 and the presence of CD44+, highly polarized, thin, and long dendrites. IFN-DCs markedly migrated in response to beta-chemokines (especially MIP-1beta) and expressed the Th-1 chemokine IP-10. Notably, IFN-DCs showed an up-regulation of CCR7 as well as of its natural ligand MIP-3beta, characteristics typical of mature DCs. Of interest, IFN-DCs exhibited a marked chemotactic response to MIP-3beta in vitro and strong migratory behavior in severe combined immunodeficient (SCID) mice. In SCID mice reconstituted with human peripheral blood leukocytes, IFN-DCs induced a potent primary human antibody response and IFN-gamma production, indicative of a Th-1 immune response. These results define the highly specialized maturation state of IFN-DCs and point out the existence of a "natural alliance" between type I IFN and monocyte/DC development, instrumental for ensuring an efficient connection between innate and adaptive immunity.

Vaginal transmission of HIV-1 in hu-SCID mice: a new model for the evaluation of vaginal microbicides.

OBJECTIVE: To develop an animal model of vaginal transmission of HIV-1 for the evaluation of vaginal microbicides. DESIGN: Vaginal infection was performed in SCID mice reconstituted with 4 x 107 human peripheral blood lymphocytes (hu-PBL) by non-invasive vaginal administration. The hu-PBL were previously infected in vitro with a non-syncytium (NSI) strain of HIV-1 (SF162) (hu-PBL-SCID). Lymphocyte migration in vivo was examined using fluorescently labelled human lymphocytes. METHODS: The percentage of CD4 T cells, plasma viral load and p24 antigen were evaluated using fluorescent activated cell sorting (FACS), the Amplicor HIV-1 monitor kit and enzyme-linked immunosorbent assay, respectively. Polymerase chain reaction (PCR) analysis was performed on DNA extracted from spleen and lymph nodes. For in vivo migration of labelled lymphocytes, the mice were sacrificed after 4, 24 and 48 h; vaginae and local lymph nodes were removed, snap frozen with OCT, sectioned and examined by fluorescent microscopy and FACS. RESULTS: HIV transmission was established using virus-infected cells inoculated vaginally, as shown by FACS, HIV viral load, p24 and PCR results. Labelled cells were easily located within the vaginal tissues after 4 h. However, few or no cells could be identified after 24 or 48 h at the vaginal level, whereas labelled cells could be detected at the level of regional lymph nodes. CONCLUSIONS: Because of its simplicity and practical features compared with other animal models, the vaginal HIV-infected hu-SCID mouse model may prove useful to test the activity of compounds against cell-associated HIV and, possibly, other sexually transmitted diseases.

HIV-1 gp120 stimulates the production of beta-chemokines in human peripheral blood monocytes through a CD4-independent mechanism.

The present study was designed to evaluate the effect of the HIV-1 envelope glycoprotein gp120 on the expression of beta-chemokines in cultured monocytes/macrophages. Treatment of either freshly isolated 1-day-cultured monocytes or 7-day-cultured monocyte-derived macrophages (MDM) with recombinant gp120-IIIB resulted in a specific and dose-dependent enhancement of secretion of monocyte chemoattractant protein-1, macrophage inflammatory protein-1beta, and RANTES as well as a clear-cut increase in transcript accumulation. The expression of these mRNA was increased, but not superinduced, in the presence of cycloheximide. beta-Chemokine secretion was also induced after exposure of monocyte cultures to gp120-JRFL and aldrithiol-2-inactivated R5 and X4 HIV-1 strains, retaining conformational and functional integrity of envelope proteins. In contrast, no beta-chemokine secretion was triggered by X4 and R5 gp120 or aldrithiol-2-inactivated virus treatment of monocytoid cell lines that were fully responsive to LPS. The gp120-mediated effect was independent of its interaction with CD4, as preincubation with soluble CD4 did not abrogate beta-chemokine induction. Moreover, triggering of CD4 receptor by a specific Ab did not result in any beta-chemokine secretion. Interestingly, engagement of CCR5 and CXCR4 receptors by specific Abs as well as treatment with CCR5 and CXCR4 ligands induced beta-chemokine secretion. On the whole, these results indicate that HIV-1 stimulates monocytes/macrophages to produce beta-chemokines by a specific interaction of gp120 with HIV-1 coreceptors on the cell membrane. The expression of these related polypeptides may represent an important cellular response for regulating both the extent of viral infection and the recruitment of immune cells.

Antitumor activity of recombinant adenoviral vectors expressing murine IFN-alpha in mice injected with metastatic IFN-resistant tumor cells.

Recent studies have shown that gene therapy with type I interferon (IFN) in an adenovirus vector is a powerful tool to suppress the growth of human tumors transplanted in immune-deficient mice. However, in these studies the host immune-mediated effects, which may be important in mediating the long-term control of tumor growth by these cytokines, was not studied. In this paper, we evaluate the antitumor efficacy of different adenoviral vectors containing mouse IFN-alpha genes (i.e., a first-generation replication-defective vector containing IFN-alpha1 and two different second-generation vectors containing IFN-alpha2) in immunocompetent DBA/2 mice transplanted with highly metastatic Friend leukemic cells resistant in vitro to type I IFN. We found that injection of all the different adenovirus vectors containing mouse IFN-alpha( genes resulted in a marked antitumor response in mice transplanted either subcutaneously or intravenously with IFN-resistant Friend leukemic cells compared to tumor-bearing animals inoculated with a control vector. Tumor growth inhibition after injection of IFN-adenovirus vectors was associated with a prolonged presence of high IFN levels in the sera of the injected mice. Suppression of metastatic tumor growth was also observed after a single injection of the IFN--adenovirus recombinant vectors, whereas a comparable antitumor response generally required several injections of high doses of IFN. Altogether, these results demonstrate that IFN--adenoviral vectors can efficiently inhibit metastatic tumor growth by host-mediated mechanisms and suggest that adenovirus-mediated IFN-alpha gene therapy may represent an attractive alternative to the conventional clinical use of this cytokine, which generally requires multiple injections of high IFN doses for a prolonged period of time.

IFN-beta stimulates the production of beta-chemokines in human peripheral blood monocytes. Importance of macrophage differentiation.

We investigated the effect of IFN-beta on beta-chemokine expression in differentiating human peripheral blood monocytes. MCP-1, MIP-1alpha and MIP-1beta were constitutively expressed in 1 day-cultured monocytes, and their secretion increased with time in culture despite any change in mRNA accumulation. IFN-beta treatment of differentiating monocytes resulted in a marked and dose-dependent increase of beta-chemokine secretion, which was regulated differently with respect to the differentiation stage. In particular, IFN-beta upregulated MCP-1 secretion in monocytes at all stages of differentiation although its effect was significantly higher in 1-day cultured monocytes as compared to monocyte-derived macrophages (MDM). In contrast, MIP-1alpha and MIP-1beta secretion was up-regulated by IFN-beta only in MDM. Although MCP-1, MIP-1alpha and MIP-1beta mRNA expression was up-regulated by IFN-beta in both 1 day-cultured monocytes and MDM, no correlation was found between mRNA level and protein secretion. These results suggest that the regulation of beta-chemokine secretion in monocytes/macrophages by IFN-beta occurred through different mechanisms, involving both a direct effect of this cytokine on chemokine gene expression and translational/post-translational steps of regulation more likely linked to the differentiation process. This finding reveals a novel role for this cytokine in the recruitment of specific cell types during the immune response, which may be relevant in the control of viral infections in vivo.

Regulation of chemokine/cytokine network during in vitro differentiation and HIV-1 infection of human monocytes: possible importance in the pathogenesis of AIDS.

The monocyte/macrophage lineage represents heterogeneous cell populations characterized by major differences in the phenotype and functional activities. These cells are a major source of soluble factors, such as cytokines and chemokines, which can both affect HIV replication and AIDS pathogenesis. Although monocytes/macrophages are unanimously considered important targets of HIV-1 infection, the HIV-induced alterations in their physiological functions at different stages of differentiation are still matter of debate. In this article, we review our data on the regulation of chemokine/cytokine network with regard to macrophage differentiation and HIV-1 infection, in comparison with studies from other groups. The ensemble of the results emphasizes that: 1) macrophages markedly differ with respect to monocytes for a variety of responses potentially important in the pathogenesis of HIV infection; and 2) the experimental conditions can influence the HIVmonocyte/macrophage interactions, reflecting the possible in vivo existence of a spectrum of responses among macrophage populations.

Dual role of the HIV-1 vpr protein in the modulation of the apoptotic response of T cells.

We investigated the effect of vpr, physiologically expressed during the course of an acute HIV-1 infection, on the response of infected cells to apoptotic stimuli as well as on the HIV-induced apoptosis. At 48 h after infection, Jurkat cells exhibited a lower susceptibility to undergo apoptosis with respect to uninfected cells. This effect was not observed following infection with either a vpr-mutated virus or a wild-type strain in the presence of antisense oligodeoxynucleotides targeted at vpr mRNA. Single-cell analysis, aimed at simultaneously identifying apoptotic and infected cells, revealed that resistance to apoptosis correlated with productive infection. Notably, vpr-dependent protection from induced apoptosis was also observed in HIV-1-infected PBMC. In contrast, at later stages of infection, a marked increase in the number of cells spontaneously undergoing apoptosis was detected in infected cultures. This virus-induced apoptosis involved vpr expression and predominantly occurred in productively infected cells. These results indicate that HIV-1 vpr can exert opposite roles in the regulation of apoptosis, which may depend on the level of its intracellular expression at different stages of HIV-1 infection. The dual function of vpr represents a novel mechanism in the complex strategy evolved by HIV to influence the turnover of T lymphocytes leading to either viral persistence or virus release and spreading.

Gene therapy of cancer with interferon: lessons from tumor models and perspectives for clinical applications.

Cytokine gene transfer is a current approach in studies of gene therapy of cancer IFNs represent valuable cytokines for these studies, since they exert multiple biological effects, including anti-tumor activities. Early studies have been focused on IFN-gamma. Recently, several reports have shown that the transfer of type I IFN (especially IFN-alpha) genes represents a powerful approach for inducing tumor suppression. Recent studies have underscored new IFN-induced activities on immune cells. This knowledge adds a further rationale for the use of IFN-alpha in strategies of gene therapy of cancer and can be exploited for the design of more selective and effective anticancer treatments.

Murine granulocytes control human tumor growth in SCID mice.

Severe combined immunodeficient (SCID) mice generally do not reject allogeneic or xenogeneic organ grafts and represent a unique model for investigating in vivo the behaviour of both normal and neoplastic human cells. However, cells from human primary tumors often do not grow in SCID mice. We have previously shown that the major reaction of SCID mice to the engraftment of human peripheral blood leukocytes is a massive granulocyte recruitment into the site of transplantation. In this study, we have investigated the role of murine granulocytes in the control of human tumor cell growth in SCID mice. We report here that murine granulocytes infiltrate and delimit the human tumor mass and that treatment of SCID mice with anti-murine granulocyte antibody markedly improves the growth of human tumor cell lines of different origin through suppression of the host granulocyte reaction. This finding provides a new tool for improving the human tumor take in SCID mice, thus opening new perspectives for a practical in vivo preclinical test of anti-tumor strategies. Moreover, this study, even with the limits of the known natural reaction against xenotransplants, further supports the importance of granulocytes in the control of tumor take and growth.

Type I interferon as a powerful adjuvant for monocyte-derived dendritic cell development and activity in vitro and in Hu-PBL-SCID mice.

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((R)) 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.

Inhibition of angiogenesis and vascular tumor growth by interferon-producing cells: A gene therapy approach.

We developed an in vivo gene therapy approach to characterize and optimize the anti-angiogenic activity of class I interferons (IFNs), using packaging cell lines producing an amphotropic LXSN-based retrovirus expressing either IFN-alpha1 (alpha1Am12), IFN-beta (betaAm12) murine cDNAs, or the vector alone (neoAm12). Pretreatment of endothelial-like Eahy926 cells in vitro with conditioned media (CM) from alpha1Am12 or betaAm12 cells for 48 hours significantly inhibited their migration and invasion as compared to neoAm12-CM-treated cells. betaAm12-CM also inhibited the formation of capillary-like structures on Matrigel by EAhy926 cells. In vivo, inclusion of the betaAm12 cells strongly inhibited, and alpha1Am12 partially inhibited, the angiogenic response in the Matrigel sponge model in both immune-competent and athymic nude mice. Electron microscopy showed a reduction of host cell infiltration in alpha1Am12- and betaAm12-containing sponges and reduction of invading tubular clefts of host cells as compared to controls. Finally, inoculation of either alpha1Am12 or betaAm12 cells (10%) along with a highly angiogenic Kaposi's sarcoma cell line (90%) resulted in a powerful reduction of tumor growth in nude mice in vivo, as did infection with the interferon-alpha-producing retroviruses. These data suggest that a gene therapy approach using class I interferons can effectively inhibit tumor angiogenesis and growth of vascular tumors.

The HIV-1 vpr protein induces anoikis-resistance by modulating cell adhesion process and microfilament system assembly.

We have previously shown that CD4+ T Jurkat cells constitutively expressing low levels of the human immunodeficiency virus 1 (HIV-1) vpr protein were less susceptible to undergo apoptosis than control cells.1 In this study we have investigated the role of vpr in affecting mechanisms of importance in the control of apoptosis. Vpr-expressing clones consistently aggregated in clusters with time in culture, whereas mock-transfected cells grew as dispersed cultures. The analysis of adhesion molecules involved in cell-to-cell as well as in cell-substrate interactions showed a higher expression of cadherin and integrins alpha5 and alpha6 in vpr-transfected clones with respect to mock-transfected cells. This up-modulation was specifically blocked by cell exposure to antisense oligonucleotides targeted at the vpr. In addition, F-actin microfilament cytoskeletal organization, known to be involved in cell-cell interaction pathways and in the modulation of cell surface molecule expression, was significantly improved in vpr-expressing clones, in which filament polymerization was increased. We thus envisage that vpr viral protein can maintain cell survival via a specific activity on cytoskeleton-dependent cell adhesion pathways, i.e. by inducing anoikis-resistance. These particular effects of vpr might enhance the homing, spreading and survival of the infected lymphocytes, thus contributing to virus persistence in the course of acute HIV-1 infection.