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Cancer stem cells (CSCs) are a subpopulation of cancer cells endowed with high tumorigenic, chemoresistant and metastatic potential. Nongenetic mechanisms of acquired resistance are increasingly being discovered, but molecular insights into the evolutionary process of CSCs are limited. Here, we show that type I interferons (IFNs-I) function as molecular hubs of resistance during immunogenic chemotherapy, triggering the epigenetic regulator demethylase 1B (KDM1B) to promote an adaptive, yet reversible, transcriptional rewiring of cancer cells towards stemness and immune escape. Accordingly, KDM1B inhibition prevents the appearance of IFN-I-induced CSCs, both in vitro and in vivo. Notably, IFN-I-induced CSCs are heterogeneous in terms of multidrug resistance, plasticity, invasiveness and immunogenicity. Moreover, in breast cancer (BC) patients receiving anthracycline-based chemotherapy, KDM1B positively correlated with CSC signatures. Our study identifies an IFN-I → KDM1B axis as a potent engine of cancer cell reprogramming, supporting KDM1B targeting as an attractive adjunctive to immunogenic drugs to prevent CSC expansion and increase the long-term benefit of therapy., (© 2022. The Author(s).)

Salivary gland tumors are a heterogeneous group of neoplasms representing less than 10% of all head and neck tumors. Among salivary gland tumors, salivary duct carcinoma (SDC) is a rare, but highly aggressive malignant tumor resembling ductal breast carcinoma. Sublingual treatments are promising for SDC due to the induction of both local and systemic biological effects and to reduced systemic toxicity compared to other administration routes. In the present study, we first established that the sublingual administration of type I IFN (IFN-I) is safe and feasible, and exerts antitumor effects both as monotherapy and in combination with chemotherapy in transplantable tumor models, i.e., B16-OVA melanoma and EG.7-OVA lymphoma. Subsequently, we proved that sublingual IFN-I in combination with cyclophosphamide (CTX) induces a long-lasting reduction of tumor mass in NeuT transgenic mice that spontaneously develop SDC. Most importantly, tumor shrinkage in NeuT transgenic micewas accompanied by the emergence of tumor-specific cellular immune responses both in the blood and in the tumor tissue. Altogether, these results provide evidence that sublingual IFN holds promise in combination with chemotherapy for the treatment of cancer.

Type I interferons (IFNs) exert anti-proliferative, antiviral and immunomodulatory activities. They are also involved in cell differentiation and anti-tumor defense processes. A growing body of literature indicates that the success of conventional chemotherapeutics, epigenetic drugs, targeted anticancer agents and radiotherapy (RT) relies, at least in part, on the induction of type I IFN signaling in malignant cells, tumor-infiltrating antigen presenting cells or other immune cells within lymphoid organs or blood. The mechanisms underlying type I IFN induction and the clinical consequences of these observations are only beginning to be elucidated. In the present manuscript, we reviewed the recent advances in the field and provided our personal view on the role of type I IFNs induced in the context of cytotoxic anticancer treatments and on its possible exploitation as a complement in cancer therapy., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Resistance to IFN-I-induced antineoplastic effects has been reported in many tumors and arises, in part, from epigenetic silencing of IFN-stimulated genes by DNA methylation. We hypothesized that restoration of IFN-stimulated genes by co-administration of the demethylating drug 5-aza-2'-deoxycitidine (decitabine [DAC]) may enhance the susceptibility to IFN-I-mediated antitumoral effects in melanoma. We show that combined administration of IFN-I and DAC significantly inhibits the growth of murine and human melanoma cells, both in vitro and in vivo. Compared with controls, DAC/IFN-I-treated melanoma cells exhibited reduced cell growth, augmented apoptosis, and diminished migration. Moreover, IFN-I and DAC synergized to suppress the growth of three-dimensional human melanoma spheroids, altering tumor architecture. These direct antitumor effects correlated with induction of the IFN-stimulated gene Mx1. In vivo, DAC/IFN-I significantly reduced melanoma growth via stimulation of adaptive immunity, promoting tumor-infiltrating CD8 + T cells while inhibiting the homing of immunosuppressive CD11b + myeloid cells and regulatory T cells. Accordingly, exposure of human melanoma cells to DAC/IFN-I induced the recruitment of immune cells toward the tumor in a Matrigel (Corning Life Sciences, Kennebunkport, ME)-based microfluidic device. Our findings underscore a beneficial effect of DAC plus IFN-I combined treatment against melanoma through both direct and immune-mediated anti-tumor effects., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Some of the anti-neoplastic effects of anthracyclines in mice originate from the induction of innate and T cell-mediated anticancer immune responses. Here we demonstrate that anthracyclines stimulate the rapid production of type I interferons (IFNs) by malignant cells after activation of the endosomal pattern recognition receptor Toll-like receptor 3 (TLR3). By binding to IFN-α and IFN-β receptors (IFNARs) on neoplastic cells, type I IFNs trigger autocrine and paracrine circuitries that result in the release of chemokine (C-X-C motif) ligand 10 (CXCL10). Tumors lacking Tlr3 or Ifnar failed to respond to chemotherapy unless type I IFN or Cxcl10, respectively, was artificially supplied. Moreover, a type I IFN-related signature predicted clinical responses to anthracycline-based chemotherapy in several independent cohorts of patients with breast carcinoma characterized by poor prognosis. Our data suggest that anthracycline-mediated immune responses mimic those induced by viral pathogens. We surmise that such 'viral mimicry' constitutes a hallmark of successful chemotherapy.

Cross-presentation is a crucial mechanism for generating CD8 T cell responses against exogenous Ags, such as dead cell-derived Ag, and is mainly fulfilled by CD8α(+) dendritic cells (DC). Apoptotic cell death occurring in steady-state conditions is largely tolerogenic, thus hampering the onset of effector CD8 T cell responses. Type I IFNs (IFN-I) have been shown to promote cross-priming of CD8 T cells against soluble or viral Ags, partly through stimulation of DC. By using UV-irradiated OVA-expressing mouse EG7 thymoma cells, we show that IFN-I promote intracellular Ag persistence in CD8α(+) DC that have engulfed apoptotic EG7 cells, regulating intracellular pH, thus enhancing cross-presentation of apoptotic EG7-derived OVA Ag by CD8α(+) DC. Notably, IFN-I also sustain the survival of Ag-bearing CD8α(+) DC by selective upmodulation of antiapoptotic genes and stimulate the activation of cross-presenting DC. The ensemble of these effects results in the induction of CD8 T cell effector response in vitro and in vivo. Overall, our data indicate that IFN-I cross-prime CD8 T cells against apoptotic cell-derived Ag both by licensing DC and by enhancing cross-presentation.

Successful chemotherapy accounts for both tumor-related factors and host immune response. Compelling evidence suggests that some chemotherapeutic agents can induce an immunogenic type of cell death stimulating tumor-specific immunity. Here, we show that cyclophosphamide (CTX) exerts two types of actions relevant for the induction of antitumor immunity in vivo: (i) effect on dendritic cell (DC) homeostasis, mediated by endogenous type I interferons (IFN-I), leading to the preferential expansion of CD8α(+) DC, the main subset involved in the cross-presentation of cell-derived antigens; and (ii) induction of tumor cell death with clear-cut immunogenic features capable of stimulating tumor infiltration, engulfment of tumor apoptotic material, and CD8 T-cell cross-priming by CD8α(+) DC. Notably, the antitumor effects of CTX were efficiently amplified by IFN-I, the former providing a source of antigen and a "resetting" of the DC compartment and the latter supplying optimal costimulation for T-cell cross-priming, resulting in the induction of a strong antitumor response and tumor rejection. These results disclose new perspectives for the development of targeted and more effective chemoimmunotherapy treatments of cancer patients.

DC are the most potent antigen-presenting cells that recognise signs of infection and serve as the main activators of naïve T cells. We have previously shown that type I IFN (IFN-I) are produced by DC and can act in an autocrine manner to activate DC. In the present study, we have investigated the role of IFN-I in regulating the turnover and lifespan of DC. We found that DC, especially the CD8alpha(+) subset, from type I IFN receptor knock out (IFNAR KO) mice, display a reduced turnover rate when compared with DC from WT mice, as revealed by BrdU labelling kinetics. In vitro, IFNAR KO BM precursor cells cultured in the presence of GM-CSF generated CD11c(+) DC less efficiently than WT BM, and the IFNAR KO DC that arose displayed reduced migratory ability. Interestingly, splenic DC from IFNAR KO mice exhibited a higher survival rate in short-term culture compared with control DC. Exposure to IFN-I in vivo markedly increased the turnover rate of splenic DC, particularly CD8alpha(+) DC, which was preceded by a transient induction of apoptosis. In accordance with this, IFN-I stimulated the apoptosis of splenic DC in vitro. Overall, our data indicate that IFN-I are important regulators of DC turnover in vivo and suggest that these cytokines may exert this function through the modulation of multiple processes involving DC apoptosis, proliferation and migration.

Presently, new attention is given to type I interferons (IFNs) as essential factors linking innate and adaptive immunity. Several studies provided evidence about the importance of IFN-alpha in the differentiation of the Th1 subset, in the generation and activity of cytotoxic T lymphocytes, in the enhancement of a primary antibody response and in the activation of dendritic cells. Owing to their immunomodulatory properties, type I IFNs can represent good candidates to be used as adjuvants for vaccination. In the present review, we summarize recent studies in humans and in animal models, suggesting a possible application of type I IFNs as adjuvants for the development of more effective vaccines against infectious diseases and cancer.

Type I IFN is a cytokine family endowed with multiple biological activities. In recent years, type I IFN has been demonstrated to play a crucial role in innate immunity, in dendritic cell maturation/differentiation and in the priming of primary antibody responses, especially when administered i.m. with a purified influenza vaccine. Due to the increasing interest in mucosal vaccination especially for respiratory infections, we investigated two different IFN-adjuvanted immunization protocols against influenza. In the first one, anesthesized C3H/HeN mice were instilled intranasally with 50 microl of a commercially available influenza vaccine containing 5 mg of hemagglutinin (HA) and a partially purified preparation of mouse IFN-alphal. A single intranasal administration of IFN-adjuvanted vaccine resulted in a full protection of 100% of mice against virus challenge while vaccine alone was only partially effective (40%). From the analysis of the specific antibody response emerged that type I IFN induced a significant increase of antibody titers in all the Ig subclasses with particular effect on IgG2a and IgA. To mimic aerosol administration and to limit vaccine delivery strictly to nasal mucosa, a second method of intranasal vaccination was developed in which mice were left awake and their nostrils moistened along the day of treatment (every 10 min) with six mini-doses (8 microl each) of vaccine +/- IFN-I with dose and timing equivalent to previous immunization protocol. This vaccination schedule prevented mice from pulmonary damage and the concomintant use of type I IFN induced an efficient and long lasting both local and systemic immune response. These findings shed new light on the involvement of type I IFN in the early phases of the immune response and open new and practical perspectives in vaccine research.

In view of the increasing interest in mucosal vaccination, we investigated whether type I IFN could act as adjuvant of an intranasally administered influenza vaccine. A single intranasal administration of IFNalphabeta-adjuvanted vaccine in anesthetized C3H/HeN mice was capable of protecting the totality of animals against virus challenge, while vaccine alone was only partially effective. To mimic intranasal vaccine administration in man and to limit vaccine delivery strictly to nasal mucosa, we used a second method of vaccination based on vaccine fractionation in six doses and intranasal instillation in non-anesthetized mice. By using this vaccination schedule, IFNalphabeta-adjuvanted vaccine also prevented mice from disease development and induced an efficient long lasting immune response. Further experiments showed that IFNalphabeta increased the percentage of antigen-associated phagocytes in the nasal mucus layer, thus suggesting a new possible mechanism of action for type I IFN as an adjuvant.

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.

In a previous study, we reported that a single injection of cyclophosphamide (CTX) in tumor-bearing mice resulted in tumor eradication when the animals were subsequently injected with tumor-sensitized lymphocytes. Notably, CTX acted by inducing bystander effects on T cells, and the response to the combined CTX/adoptive immunotherapy regimen was inhibited in mice treated with antibodies to mouse interferon (IFN)-alpha/beta. In the present study, we have investigated whether CTX induced the expression of type I IFN, and we have characterized the CTX effects on the phenotype of T cells in normal mice. CTX injection resulted in an accumulation of type I IFN messenger RNA in the spleen of inoculated mice, at 24 to 48 hours, that was associated with IFN detection in the majority of the animals. CTX also enhanced the expression of the Ly-6C on spleen lymphocytes. This enhancement was inhibited in mice treated with anti-type I IFN antibodies. Moreover, CTX induced a long-lasting increase in in vivo lymphocyte proliferation and in the percentage of CD44(hi)CD4(+) and CD44(hi)CD8(+ )T lymphocytes. These results demonstrate that CTX is an inducer of type I IFN in vivo and enhances the number of T cells exhibiting the CD44(hi) memory phenotype. Since type I IFN has been recently recognized as the important cytokine for the in vivo expansion and long-term survival of memory T cells, we suggest that induction of this cytokine may explain at least part of the immunomodulatory effects observed after CTX treatment. Finally, these findings provide a new rationale for combined treatments with CTX and adoptive immunotherapy in cancer patients. (Blood. 2000;95:2024-2030)