Type I IFNs promote cancer cell stemness by triggering the epigenetic regulator KDM1B.

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.

Immunomodulatory properties of CNF1 toxin from E. coli: implications for colorectal carcinogenesis.

Colorectal cancer (CRC) is a leading cause of cancer death worldwide. The risk of developing CRC is influenced by both environmental and genetic factors. Recently, chronic inflammation and gut microbiota modifications have been associated with increased CRC risk. Escherichia coli belongs to the commensal intestinal flora and can become highly pathogenic following the acquisition of genes coding for virulence factors, such as the cytotoxic necrotizing factor type 1 (CNF1). Numerous reports highlight that, besides exerting direct effects on epithelial cells, CNF1 can also act on immune cells, modulating their responses and possibly contributing to disease development. In the present review, we summarized the key studies addressing the immunomodulatory functions of CNF1 and discussed the contribution that CNF1 can bring about to CRC through the creation of a pro-inflammatory microenvironment.

Exploiting natural antiviral immunity for the control of pandemics: Lessons from Covid-19.

The outbreak of coronavirus disease 2019 (COVID-19), triggered by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and the disruptive global consequences in terms of mortality and social and economic crises, have taught lessons that may help define strategies to better face future pandemics. Innate and intrinsic immunity form the front-line natural antiviral defense. They involve both tissue-resident and circulating cells, which can produce anti-viral molecules shortly after viral infection. Prototypes of these factors are type I interferons (IFN), antiviral cytokines with a long record of clinical use. During the last two years, there has been an impressive progress in understanding the mechanisms of both SARS-CoV-2 infection and the cellular and soluble antiviral responses occurring early after viral exposure. However, this information was not sufficiently translated into therapeutic approaches. Insufficient type I IFN activity probably accounts for disease progression in many patients. This results from both the multiple interfering mechanisms developed by SARS-CoV-2 to decrease type I IFN response and various pre-existing human deficits of type I IFN activity, inherited or auto-immune. Emerging data suggest that IFN-I-mediated boosting of patients' immunity, achieved directly through the exogenous administration of IFN-ß early post viral infection, or indirectly following inoculation of heterologous vaccines (e.g., Bacillus Calmette Guerin), might play a role against SARS-CoV-2. We review how recent insights on the viral and human determinants of critical COVID-19 pneumonia can foster clinical studies of IFN therapy. We also discuss how early therapeutic use of IFN-ß and prophylactic campaigns with live attenuated vaccines might prevent a first wave of new pandemic viruses.

Dietary Polyphenols: Promising Adjuvants for Colorectal Cancer Therapies.

Colorectal cancer (CRC) is a major cancer type and a leading cause of death worldwide. Despite advances in therapeutic management, the current medical treatments are not sufficient to control metastatic disease. Treatment-related adverse effects and drug resistance strongly contribute to therapy failure and tumor recurrence. Combination therapy, involving cytotoxic treatments and non-toxic natural compounds, is arousing great interest as a promising more effective and safer alternative. Polyphenols, a heterogeneous group of bioactive dietary compounds mainly found in fruit and vegetables, have received great attention for their capacity to modulate various molecular pathways active in cancer cells and to affect host anticancer response. This review provides a summary of the most recent (i.e., since 2016) preclinical and clinical studies using polyphenols as adjuvants for CRC therapies. These studies highlight the beneficial effects of dietary polyphenols in combination with cytotoxic drugs or irradiation on both therapy outcome and drug resistance. Despite substantial preclinical evidence, data from a few pilot clinical trials are available to date with promising but still inconclusive results. Larger randomized controlled studies and polyphenol formulations with improved bioavailability are needed to translate the research progress into clinical applications and definitively prove the added value of these molecules in CRC management.

Antiviral and immunomodulatory interferon-beta in high-risk COVID-19 patients: a structured summary of a study protocol for a randomised controlled trial.

OBJECTIVES: The primary objective of the study is to demonstrate the efficacy of low-dose IFN-ß in reducing the risk of SARS-CoV-2 recently infected elderly patients to progress towards severe COVID-19 versus control group within 28 days. Secondary objectives are: 1) To assess the reduction in Intensive Care Unit (ICU) admission in patients treated with IFN-ß versus control group within 28 days of randomization 2) To assess the reduction in number of deaths in IFN- ß compared to control group (day 28) 3) To evaluate the increase in proportion of participants returning to negative SARS-CoV-2 RT-PCR in IFN-ß -treated versus control group at Day 14 and Day 28 4) To assess the increase in SARS-CoV-2-specific binding antibody titers in IFN-ß compared to control group (day 28) 5) To assess the safety of IFN-ß -treated patients versus control group TRIAL DESIGN: Randomized, Open-Label, Controlled, Superiority Phase II Study. Patients, who satisfy all inclusion criteria and no exclusion criteria, will be randomly assigned to one of the two treatment groups in a ratio 2:1 (IFN-treated versus control patients). Randomization will be stratified by gender. Stratified randomization will balance the presence of male and female in both study arms. PARTICIPANTS: Male and female adults aged 65 years or older with newly diagnosed SARS-CoV-2 infection and mild COVID-19 symptoms are eligible for the study. The trial is being conducted in Rome. Participants will be either hospitalized or home isolated. A group of physicians belonging to the Special Unit for Regional Continued Care (USCAR), specifically trained for the study and under the supervision of the National Institute for Infectious Diseases "Lazzaro Spallanzani", will be responsible for the screening, enrolment, treatment and clinical monitoring of patients, thus acting as a bridge between clinical centers and territorial health management. Inclusion criteria are as follows: ≥ 65 years of age at time of enrolment; Laboratory-confirmed SARS-CoV-2 infection as determined by PCR, in any specimen < 72 hours prior to randomization; Subject (or legally authorized representative) provides written informed consent prior to initiation of any study procedures; Understands and agrees to comply with planned study procedures; Agrees to the collection of nasopharyngeal swabs and venous blood samples per protocol; Being symptomatic for less than 7 days before starting therapy; NEWS2 score ≤2. Exclusion criteria are as follows: Hospitalized patients with illness of any duration, and at least one of the following: Clinical assessment (evidence of rales/crackles on exam) and SpO2 ≤ 94% on room air at rest or after walking test, OR Acute respiratory failure requiring mechanical ventilation and/or supplemental oxygen; Patients currently using IFN-ß (e.g., multiple sclerosis patients); Patients undergoing chemotherapy or other immunosuppressive treatments; Patients with chronic kidney diseases; Known allergy or hypersensitivity to IFN (including asthma); Any autoimmune disease (resulting from patient anamnesis); Patients with signs of dementia or neurocognitive disorders; Patients with current severe depression and/or suicidal ideations; Being concurrently involved in another clinical trial; HIV infection (based on the anamnesis); Use of any antiretroviral medication; Impaired renal function (eGFR calculated by CKD-EPI Creatinine equation < 30 ml/min); Presence of other severe diseases impairing life expectancy (e.g. patients are not expected to survive 28 days given their pre-existing medical condition); Any physical or psychological impediment in a patient that could let the investigator to suspect his/her poor compliance; Lack or withdrawal of informed consent INTERVENTION AND COMPARATOR: Control arm: No specific antiviral treatment besides standard of care. Treatment arm: 11µg (3MIU) of IFN-ß1a will be injected subcutaneously at day 1, 3, 7, and 10 in addition to standard of care. The drug solution, contained in a pre-filled cartridge, will be injected by means of the RebiSmart® electronic injection device. Interferon ß1a (Rebif®, Merck KGaA, Darmstadt, Germany) is a disease-modifying drug used to treat relapsing forms of multiple sclerosis (MS). The dose selected for this study is expected to exploit the antiviral and immunomodulatory properties of the cytokine without causing relevant toxicity or inducing refractoriness phenomena sometimes observed after high-dose and/or chronic IFNß treatments. MAIN OUTCOMES: Primary endpoint of the study is the proportion of patients experiencing a disease progression, during at least 5 days, according to the National Early Warning Score (NEWS2). The NEWS2 score is a standardized approach aimed at promptly detecting signs of clinical deterioration in acutely ill patients and establishing the potential need for higher level of care. It is based on the evaluation of vital signs, including respiratory rate, oxygen saturation, temperature, blood pressure, pulse/heart rate, AVPU response. The resulting observations, compared to a normal range, are combined in a single composite "alarm" score. Any other clinical sign clearly indicating a disease worsening will be considered as disease progression. RANDOMIZATION: Sixty patients will be randomized 2:1 to receive IFN-ß1a plus the standard of care or the standard of care only. Eligible patients will be randomized (no later than 36 h after enrolment) by means of a computerized central randomization system. All patients will receive a unique patient identification number at enrolling visit when signing the informed consent and before any study procedure is performed. This number remains constant throughout the entire study. The randomization of patients will be closed when 60 patients have been enrolled. The randomization will be stratified by sex; for each stratum a sequence of treatments randomly permuted in blocks of variable length (3 or 6) will be generated. BLINDING (MASKING): This is an open-label study. After the randomization, patients will be notified whether they will be in the experimental arm or in the control arm. NUMBERS TO BE RANDOMISED (SAMPLE SIZE): The study plans to enrol 60 patients: 40 in the IFN-ß1a arm, 20 in the control arm, according to a 2:1 - treated: untreated ratio. TRIAL STATUS: Protocol Version: 3.0 Version Date: 18/03/2021 The study is open for recruitment since 16/04/2021.Recruitment is expected to l be completed before 15/08/2021. TRIAL REGISTRATION: EudraCT N°: 2020-003872-42, registration date: 19/10/2020. FULL PROTOCOL: The full protocol is attached as an additional file, accessible from the Trials website (Additional file 1). In the interest in expediting dissemination of this material, the familiar formatting has been eliminated; this Letter serves as a summary of the key elements of the full protocol."

Anticancer Effects of Sublingual Type I IFN in Combination with Chemotherapy in Implantable and Spontaneous Tumor Models.

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.

Towards a Systems Immunology Approach to Unravel Responses to Cancer Immunotherapy.

Immunotherapy, particularly immune checkpoint blockade and chimeric antigen receptor (CAR)-T cells, holds a great promise against cancer. These treatments have markedly improved survival in solid as well as in hematologic tumors previously considered incurable. However, durable responses occur in a fraction of patients, and existing biomarkers (e.g. PD-L1) have shown limited prediction power. This scenario highlights the need to dissect the complex interplay between immune and tumor cells to identify reliable biomarkers of response to be used for patients' selection. In this context, systems immunology represents indeed the new frontier to address important clinical challenges in biomarker discovery. Through the integration of multiple layers of data obtained with several high-throughput approaches, systems immunology may give insights on the vast range of inter-individual differences and on the influences of genes and factors that cooperatively shape the individual immune response to a given treatment. In this Mini Review, we give an overview of the current high-throughput methodologies, including genomics, epigenomics, transcriptomics, metabolomics, proteomics, and multi-parametric phenotyping suitable for systems immunology as well as on the key steps of data integration and biological interpretation. Additionally, we review recent studies in which multi-omics technologies have been used to characterize mechanisms of response and to identify powerful biomarkers of response to checkpoint inhibitors, CAR-T cell therapy, dendritic cell-based and peptide-based cancer vaccines. We also highlight the need of favoring the collaboration of researchers with complementary expertise and of integrating multi-omics data into biological networks with the final goal of developing accurate markers of therapeutic response.

Are we fully exploiting type I Interferons in today's fight against COVID-19 pandemic?

Coronavirus disease 2019 (COVID-19) first emerged in late 2019 in China. At the time of writing, its causative agent SARS-CoV-2 has spread worldwide infecting over 9 million individuals and causing more than 460,000 deaths. In the absence of vaccines, we are facing the dramatic challenge of controlling COVID-19 pandemic. Among currently available drugs, type I Interferons (IFN-I) - mainly IFN-α and ß -represent ideal candidates given their direct and immune-mediated antiviral effects and the long record of clinical use. However, the best modalities of using these cytokines in SARS-CoV-2 infected patients is a matter of debate. Here, we discuss how we can exploit the current knowledge on IFN-I system to tailor the most promising dosing, timing and route of administration of IFN-I to the disease stage, with the final aim of making these cytokines a valuable therapeutic strategy in today's fight against COVID-19 pandemic.

Tumor-Intrinsic or Drug-Induced Immunogenicity Dictates the Therapeutic Success of the PD1/PDL Axis Blockade.

Immunotherapy with immune checkpoint inhibitors (ICIs) has revolutionized cancer treatment providing unprecedented clinical benefits. However, many patients do not respond to ICIs as monotherapy or develop resistance. Combining ICI-based immunotherapy with chemotherapy is a promising strategy to increase response rates, but few rationale-driven chemo-immunotherapy combinations have reached the clinical arena thus far. In the present study, we show that combined anti-PDL1 and anti-PDL2 antibodies optimally synergize with cyclophosphamide but not with cisplatin, and that the magnitude and duration of the therapeutic response is dependent on the immunogenic potential of the drug and of the tumor itself. Hallmarks of successful therapeutic outcomes were the enhanced infiltration by myeloid (mainly cross-presenting dendritic cells, eosinophils, and monocytic myeloid cells) and T lymphocytes into the tumor tissue and the expansion of circulating memory pools. Overall, our results suggest that immunomodulating chemotherapy can be exploited to increase the efficacy of PD1/PDL axis inhibitors in vivo, and that the magnitude of the synergic therapeutic response is affected by tumor-intrinsic immunogenicity.

Enzyme-linked immunospot assay to monitor antigen-specific cellular immune responses in mouse tumor models.

Critical to the advancement of tumor immunotherapy is the reliable identification of responders and the quantification of the tumor-specific immune response elicited by treatments. In this regard, Enzyme-Linked Immunospot assay (ELISpot) is an ideal monitoring technique due to its high sensitivity, ease of execution and cost-effectiveness. Originally developed for the enumeration of B cells secreting antigen-specific antibodies, ELISpot assay has been adapted to detect and quantify cytokine-secreting immune cells present at low frequency in a variety of biological samples, including blood, in response to antigen-specific stimuli. The above-mentioned features emphasize the role of ELISpot as valuable assay for translational research and clinical applications. In the present chapter, we will focus on the use of ELISpot assay for monitoring the tumor-specific effector responses induced by different treatments in preclinical models and will provide some protocols and technical hints for its application.

The role of exosomes in colorectal cancer disease progression and response to therapy.

Colorectal cancer (CRC) is the second leading cause of cancer mortality in both men and women worldwide. Survival of patients is significantly associated with disease stage at diagnosis. Recent studies highlighted a role of exosomes in CRC development and progression, thus raising the interest on these nanosized vesicular structures as possible biomarkers. Exosomes contain a large variety of molecules, including proteins, lipids and nucleic acids, that are exchanged between cells either within tumor microenvironment or at distant sites from the primary tumor, where they prepare a suitable soil for tumor metastases. The present review summarizes the principal effects of exosomes on CRC development, progression, and provides an update of the most recent findings on the use of exosomal molecules as diagnostic, prognostic and predictive biomarkers in CRC.

The added value of type I interferons to cytotoxic treatments 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.

Combining Type I Interferons and 5-Aza-2'-Deoxycitidine to Improve Anti-Tumor Response against Melanoma.

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.

Classification of current anticancer immunotherapies.

During the past decades, anticancer immunotherapy has evolved from a promising therapeutic option to a robust clinical reality. Many immunotherapeutic regimens are now approved by the US Food and Drug Administration and the European Medicines Agency for use in cancer patients, and many others are being investigated as standalone therapeutic interventions or combined with conventional treatments in clinical studies. Immunotherapies may be subdivided into "passive" and "active" based on their ability to engage the host immune system against cancer. Since the anticancer activity of most passive immunotherapeutics (including tumor-targeting monoclonal antibodies) also relies on the host immune system, this classification does not properly reflect the complexity of the drug-host-tumor interaction. Alternatively, anticancer immunotherapeutics can be classified according to their antigen specificity. While some immunotherapies specifically target one (or a few) defined tumor-associated antigen(s), others operate in a relatively non-specific manner and boost natural or therapy-elicited anticancer immune responses of unknown and often broad specificity. Here, we propose a critical, integrated classification of anticancer immunotherapies and discuss the clinical relevance of these approaches.

Cancer cell-autonomous contribution of type I interferon signaling to the efficacy of chemotherapy.

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-ß 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.

Consensus guidelines for the detection of immunogenic cell death.

Apoptotic cells have long been considered as intrinsically tolerogenic or unable to elicit immune responses specific for dead cell-associated antigens. However, multiple stimuli can trigger a functionally peculiar type of apoptotic demise that does not go unnoticed by the adaptive arm of the immune system, which we named "immunogenic cell death" (ICD). ICD is preceded or accompanied by the emission of a series of immunostimulatory damage-associated molecular patterns (DAMPs) in a precise spatiotemporal configuration. Several anticancer agents that have been successfully employed in the clinic for decades, including various chemotherapeutics and radiotherapy, can elicit ICD. Moreover, defects in the components that underlie the capacity of the immune system to perceive cell death as immunogenic negatively influence disease outcome among cancer patients treated with ICD inducers. Thus, ICD has profound clinical and therapeutic implications. Unfortunately, the gold-standard approach to detect ICD relies on vaccination experiments involving immunocompetent murine models and syngeneic cancer cells, an approach that is incompatible with large screening campaigns. Here, we outline strategies conceived to detect surrogate markers of ICD in vitro and to screen large chemical libraries for putative ICD inducers, based on a high-content, high-throughput platform that we recently developed. Such a platform allows for the detection of multiple DAMPs, like cell surface-exposed calreticulin, extracellular ATP and high mobility group box 1 (HMGB1), and/or the processes that underlie their emission, such as endoplasmic reticulum stress, autophagy and necrotic plasma membrane permeabilization. We surmise that this technology will facilitate the development of next-generation anticancer regimens, which kill malignant cells and simultaneously convert them into a cancer-specific therapeutic vaccine.

Exploiting dendritic cells in the development of cancer vaccines.

Due to their central role in priming and modulating the immune response, dendritic cells (DCs) represent an ideal instrument for the design of effective immunotherapeutic strategies for cancer patients. Recent advancement on the knowledge of the numerous DC subtypes, their functions and T-cell polarizing abilities has led to the development of several protocols for the ex vivo differentiation of autologous DCs and their loading with tumor-associated antigens. Moreover, novel strategies for the in vivo targeting of tumor antigens and adjuvants to natural DC subsets have been developed. Despite the large number of clinical studies carried out in cancer patients, a consensus on the optimal treatment modalities has not been reached yet. In this review, we summarize our current knowledge on DC biology and on DC use in clinical trials. Special attention is given to the many open issues regarding DC-based vaccination to sensitize researchers in the field to the compelling need of conducting comparative studies systematically addressing the still unresolved problems.

Immunomodulatory effects of cyclophosphamide and implementations for vaccine design.

Drug repositioning refers to the utilization of a known compound in a novel indication underscoring a new mode of action that predicts innovative therapeutic options. Since 1959, alkylating agents, such as the lead compound cyclophosphamide (CTX), have always been conceived, at high dosages, as potent cytotoxic and lymphoablative drugs, indispensable for dose intensity and immunosuppressive regimen in the oncological and internal medicine armamentarium. However, more recent work highlighted the immunostimulatory and/or antiangiogenic effects of low dosing CTX (also called "metronomic CTX") opening up novel indications in the field of cancer immunotherapy. CTX markedly influences dendritic cell homeostasis and promotes IFN type I secretion, contributing to the induction of antitumor cytotoxic T lymphocytes and/or the proliferation of adoptively transferred T cells, to the polarization of CD4(+) T cells into TH1 and/or TH17 lymphocytes eventually affecting the Treg/Teffector ratio in favor of tumor regression. Moreover, CTX has intrinsic "pro-immunogenic" activities on tumor cells, inducing the hallmarks of immunogenic cell death on a variety of tumor types. Fifty years after its Food and Drug Administration approval, CTX remains a safe and affordable compound endowed with multifaceted properties and plethora of clinical indications. Here we review its immunomodulatory effects and advocate why low dosing CTX could be successfully combined to new-generation cancer vaccines.

Type I IFNs control antigen retention and survival of CD8α(+) dendritic cells after uptake of tumor apoptotic cells leading to cross-priming.

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.

Cyclophosphamide synergizes with type I interferons through systemic dendritic cell reactivation and induction of immunogenic tumor apoptosis.

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.