Filippo Belardelli

Type i interferons potently enhance humoral immunity and can promote isotype switching by stimulating dendritic cells in vivo.

Type I interferons (IFN-I) are rapidly induced following infection and play a key role in nonspecific inhibition of virus replication. Here we have investigated the effects of IFN-I on the generation of antigen-specific antibody responses. The data show that IFN-I potently enhance the primary antibody response to a soluble protein, stimulating the production of all subclasses of IgG, and induce long-lived antibody production and immunological memory. In addition, endogenous production of IFN-I was shown to be essential for the adjuvant activity of CFA. Finally, IFN-I enhanced the antibody response and induced isotype switching when dendritic cells were the only cell type responding to IFN-I. The data reveal the potent adjuvant activity of IFN-I and their important role in linking innate and adaptive immunity.

IL-15 Is Expressed by Dendritic Cells in Response to Type I IFN, Double-Stranded RNA, or Lipopolysaccharide and Promotes Dendritic Cell Activation

Cytokines that are induced by infection may contribute to the initiation of immune responses through their ability to stimulate dendritic cells (DCs). In this paper, we have addressed the role of IL-15 in DC activation, investigating its expression by DCs in response to three different signals of infection and examining its ability to stimulate DCs. We report that the expression of both IL-15 and the IL-15 receptor α-chain are increased in splenic DCs from mice inoculated with dsRNA (poly(I:C)), LPS, or IFN-αβ, and in purified murine splenic DCs treated with IFN-αβ in vitro. Furthermore, IL-15 itself was able to activate DCs, as in vivo or in vitro exposure of splenic DCs to IL-15 resulted in an up-regulation of costimulatory molecules, markedly increased production of IFN-γ by DC and an enhanced ability of DCs to stimulate Ag-specific CD8+ T cell proliferation. The magnitude of all of the IL-15-induced changes in DCs was reduced in mice deficient for the IFN-αβ receptor, suggesting a role for IFN-αβ in the stimulation of DCs by IL-15. These results identify IL-15 as a stimulatory cytokine for DCs with the potential for autocrine activity and link its effects to expression of IFN-αβ.

The neglected role of type I interferon in the T-cell response: implications for its clinical use

Originally described as an antiviral substance, type I interferon (IFN) was subsequently shown to exert multiple biological effects and is now the most frequently used cytokine in the treatment of some viral and neoplastic diseases. Although early studies described various effects on the immune system, the role of type I IFN as an immunoregulatory molecule has long been neglected. Here, Filippo Belardelli and Ion Gresser summarize recent experimental results on the interactions of type I IFN with T cells, which may prove important in its use in patients.

ICSBP is essential for the development of mouse type I interferon-producing cells and for the generation and activation of CD8α+ dendritic cells

Interferon (IFN) consensus sequence-binding protein (ICSBP) is a transcription factor playing a critical role in the regulation of lineage commitment, especially in myeloid cell differentiation. In this study, we have characterized the phenotype and activation pattern of subsets of dendritic cells (DCs) in ICSBP−/− mice. Remarkably, the recently identified mouse IFN-producing cells (mIPCs) were absent in all lymphoid organs from ICSBP−/− mice, as revealed by lack of CD11clowB220+Ly6C+CD11b− cells. In parallel, CD11c+ cells isolated from ICSBP−/− spleens were unable to produce type I IFNs in response to viral stimulation. ICSBP−/− mice also displayed a marked reduction of the DC subset expressing the CD8α marker (CD8α+ DCs) in spleen, lymph nodes, and thymus. Moreover, ICSBP−/− CD8α+ DCs exhibited a markedly impaired phenotype when compared with WT DCs. They expressed very low levels of costimulatory molecules (intercellular adhesion molecule [ICAM]-1, CD40, CD80, CD86) and of the T cell area-homing chemokine receptor CCR7, whereas they showed higher levels of CCR2 and CCR6, as revealed by reverse transcription PCR. In addition, these cells were unable to undergo full phenotypic activation upon in vitro culture in presence of maturation stimuli such as lipopolysaccharide or poly (I:C), which paralleled with lack of Toll-like receptor (TLR)3 mRNA expression. Finally, cytokine expression pattern was also altered in ICSBP−/− DCs, as they did not express interleukin (IL)-12p40 or IL-15, but they displayed detectable IL-4 mRNA levels. On the whole, these results indicate that ICSBP is a crucial factor in the regulation of two possibly linked processes: (a) the development and activity of mIPCs, whose lack in ICSBP−/− mice may explain their high susceptibility to virus infections; (b) the generation and activation of CD8α+ DCs, whose impairment in ICSBP−/− mice can be responsible for the defective generation of a Th1 type of immune response.

Interferon-alpha in tumor immunity and immunotherapy.

Interferon-alpha (IFN-alpha) is a pleiotropic cytokine belonging to type I IFN, currently used in cancer patients. Early studies in mouse tumor models have shown the importance of host immune mechanisms in the generation of a long-lasting antitumor response to type I IFN. Recent studies have underscored new immunomodulatory effects of IFN-alpha, including activities on T and dendritic cells, which may explain IFN-induced tumor immunity. Reports on new immune correlates in cancer patients responding to IFN-alpha represent additional evidence on the importance of the interactions of IFN-alpha with the immune system for the generation of durable antitumor response. This knowledge, together with results from studies on genetically modified tumor cells expressing IFN-alpha, suggest novel strategies for using these cytokines in cancer immunotherapy and in particular the use of IFN-alpha as an immune adjuvant for the development of cancer 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.

A Contribution of Mouse Dendritic Cell–Derived IL-2 for NK Cell Activation

Dendritic cells (DCs) play a predominant role in activation of natural killer (NK) cells that exert their functions against pathogen-infected and tumor cells. Here, we used a murine model to investigate the molecular mechanisms responsible for this process. Two soluble molecules produced by bacterially activated myeloid DCs are required for optimal priming of NK cells. Type I interferons (IFNs) promote the cytotoxic functions of NK cells. IL-2 is necessary both in vitro and in vivo for the efficient production of IFNγ, which has an important antimetastatic and antibacterial function. These findings provide new information about the mechanisms that mediate DC–NK cell interactions and define a novel and fundamental role for IL-2 in innate immunity.

Type I IFN as a Natural Adjuvant for a Protective Immune Response: Lessons from the Influenza Vaccine Model

The identification of natural adjuvants capable of selectively promoting an efficient immune response against infectious agents would represent an important advance in immunology, with direct implications for vaccine development, whose progress is generally hampered by the difficulties in defining powerful synthetic adjuvants suitable for clinical use. Here, we demonstrate that endogenous type I IFN is necessary for the Th1 type of immune response induced by typical adjuvants in mice and that IFN itself is an unexpectedly powerful adjuvant when administered with the human influenza vaccine, for inducing IgG2a and IgA production and conferring protection from virus challenge. The finding that these cytokines, currently used in patients, are necessary for full expression of adjuvant activity and are sufficient for the generation of a protective immune response opens new perspectives in understanding the basis of immunity and in vaccine development.

Reciprocal activating interaction between dendritic cells and pamidronate-stimulated γδ T cells: Role of CD86 and inflammatory cytokines

We investigated the interactions between human monocyte-derived dendritic cells (DCs) and Ag-activated circulating TCR-γδ-expressing lymphocytes (Vδ2). Coculture of immature DCs (iDCs) with peripheral blood Vδ2 T cells activated with either pyrophosphomonoesters (isopentenyl pyrophosphate; IPP) or aminobiphosphonates (pamidronate; PAM) led to a significant up-modulation of CD86 and MHC class I molecules and to the acquisition of functional features typical of activated DCs. DC activation induced by both IPP- and PAM-stimulated γδ T cells was mostly mediated by TNF-α and IFN-γ secreted by activated lymphocytes. However, the effect of PAM-activated γδ T cells, but not that of IPP-activated cells, required cell-to-cell contact. Reciprocally, activation of Vδ2 T cells by PAM, but not by IPP, was dependent on cell contact with iDCs. In fact, when PAM-stimulated DC-γδ T cell cocultures were separated by a semipermeable membrane or treated with blocking anti-CD86 Abs, induction of CD25 and CD69 as well as IFN-γ and TNF-α secretion by Vδ2 cells were strongly reduced. These results demonstrate for the first time a bidirectional activating interaction between iDCs and PAM-stimulated γδ T lymphocytes, thus suggesting a potential adjuvant role of this early cross-talk in the therapeutic activity of aminobiphosphonate drugs.

Cyclophosphamide Enhances the Antitumor Efficacy of Adoptively Transferred Immune Cells through the Induction of Cytokine Expression, B-Cell and T-Cell Homeostatic Proliferation, and Specific Tumor Infiltration

Purpose: Immunotherapy is a promising antitumor strategy, which can be successfully combined with current anticancer treatments, as suggested by recent studies showing the paradoxical chemotherapy-induced enhancement of the immune response. The purpose of the present work is to dissect the biological events induced by chemotherapy that cooperate with immunotherapy in the success of the combined treatment against cancer. In particular, we focused on the following: (a) cyclophosphamide-induced modulation of several cytokines, (b) homeostatic proliferation of adoptively transferred lymphocytes, and (c) homing of transferred lymphocytes to secondary lymphoid organs and tumor mass. Experimental Design: Here, we used the adoptive transfer of tumor-immune cells after cyclophosphamide treatment of tumor-bearing mice as a model to elucidate the mechanisms by which cyclophosphamide can render the immune lymphocytes competent to induce tumor rejection. Results: The transfer of antitumor immunity was found to be dependent on CD4+ T cells and on the cooperation of adoptively transferred cells with the host immune system. Of note, tumor-immune lymphocytes migrated specifically to the tumor only in mice pretreated with cyclophosphamide. Cyclophosphamide treatment also promoted homeostatic proliferation/activation of transferred B and T lymphocytes. Optimal therapeutic responses to the transfer of immune cells were associated with the cyclophosphamide-mediated induction of a “cytokine storm” [including granulocyte macrophage colony-stimulating factor, interleukin (IL)-1β, IL-7, IL-15, IL-2, IL-21, and IFN-γ], occurring during the “rebound phase” after drug-induced lymphodepletion. Conclusions: The ensemble of these data provides a new rationale for combining immunotherapy and chemotherapy to induce an effective antitumor response in cancer patients.