Blood immune cells as potential biomarkers predicting relapse-free survival of stage III/IV resected melanoma patients treated with peptide-based vaccination and interferon-alpha.

Introduction: Despite the recent approval of several therapies in the adjuvant setting of melanoma, tumor relapse still occurs in a significant number of completely resected stage III-IV patients. In this context, the use of cancer vaccines is still relevant and may increase the response to immune checkpoint inhibitors. We previously demonstrated safety, immunogenicity and preliminary evidence of clinical efficacy in stage III/IV resected melanoma patients subjected to a combination therapy based on peptide vaccination together with intermittent low-dose interferon-α2b, with or without dacarbazine preconditioning (https://www.clinicaltrialsregister.eu/ctr-search/search, identifier: 2008-008211-26). In this setting, we then focused on pre-treatment patient immune status to highlight possible factors associated with clinical outcome. Methods: Multiparametric flow cytometry was used to identify baseline immune profiles in patients' peripheral blood mononuclear cells and correlation with the patient clinical outcome. Receiver operating characteristic curve, Kaplan-Meier survival and principal component analyses were used to evaluate the predictive power of the identified markers. Results: We identified 12 different circulating T and NK cell subsets with significant (p ≤ 0.05) differential baseline levels in patients who later relapsed with respect to patients who remained free of disease. All 12 parameters showed a good prognostic accuracy (AUC>0.7, p ≤ 0.05) and 11 of them significantly predicted the relapse-free survival. Remarkably, 3 classifiers also predicted the overall survival. Focusing on immune cell subsets that can be analyzed through simple surface staining, three subsets were identified, namely regulatory T cells, CD56dimCD16- NK cells and central memory γδ T cells. Each subset showed an AUC>0.8 and principal component analysis significantly grouped relapsing and non-relapsing patients (p=0.034). These three subsets were used to calculate a combination score that was able to perfectly distinguish relapsing and non-relapsing patients (AUC=1; p=0). Noticeably, patients with a combined score ≥2 demonstrated a strong advantage in both relapse-free (p=0.002) and overall (p=0.011) survival as compared to patients with a score <2. Discussion: Predictive markers may be used to guide patient selection for personalized therapies and/or improve follow-up strategies. This study provides preliminary evidence on the identification of peripheral blood immune biomarkers potentially capable of predicting the clinical response to combined vaccine-based adjuvant therapies in melanoma.

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.

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.

Type I Interferons as Joint Regulators of Tumor Growth and Obesity.

Type I interferons (IFN-I) are antiviral cytokines endowed with multiple biological actions, including antitumor activity. Studies in mouse models and cancer patients support the concept that endogenous IFN-I play important roles in the control of tumor development and growth as well as in response to several chemotherapy/radiotherapy treatments. While IFN-I signatures in the tumor microenvironment are often considered as biomarkers for a good prognostic response to antitumor therapies, prolonged IFN-I signaling can lead to immune dysfunction, thereby promoting pathogen or tumor persistence, thus revealing the "Janus face" of these cytokines in cancer control, likely depending on timing, tissue microenvironment and cumulative levels of IFN-I signals. Likewise, IFN-I exhibit different and even opposite effects on obesity, a pathologic condition linked to cancer development and growth. As an example, evidence obtained in mouse models shows that localized expression of IFN-I in the adipose tissue results in inhibition of diet-induced obesity, while hyper-production of these cytokines by specialized cells such as plasmacytoid dendritic cells in the same tissue, can induce systemic inflammatory responses leading to obesity. Further studies in mouse models and humans should reveal the mechanisms by which IFN-I can regulate both tumor growth and obesity and to understand the role of factors such as genetic background, diet and microbioma in shaping the production and action of these cytokines under physiological and pathological conditions.

Type I interferons induce peripheral T regulatory cell differentiation under tolerogenic conditions.

The type I interferons are central to a vast array of immunological functions. The production of these immune-modulatory molecules is initiated at the early stages of the innate immune responses and, therefore, plays a dominant role in shaping downstream events in both innate and adaptive immunity. Indeed, the major role of IFN-α/ß is the induction of priming states, relevant for the functional differentiation of T lymphocyte subsets. Among T-cell subtypes, the CD4+CD25+Foxp3+ T regulatory cells (Tregs) represent a specialized subset of CD4+ T cells with a critical role in maintaining peripheral tolerance and immune homeostasis. Although the role of type I interferons in maintaining the function of thymus-derived Tregs has been previously described, the direct contribution of these innate factors to peripheral Treg (pTreg) and induced Treg (iTreg) differentiation and suppressive function is still unclear. We now show that, under tolerogenic conditions, IFN-α/ß play a critical role in antigen-specific and also polyclonal naive CD4+ T-cell conversion into peripheral antigen-specific CD4+CD25+Foxp3+ Tregs and inhibit CD4+ T helper (Th) cell expansion in mice. While type I interferons sustain the expression and the activation of the transcription master regulators Foxp3, Stat3 and Stat5, these innate molecules reciprocally inhibit Th17 cell differentiation. Altogether, these results indicate a new pivotal role of IFN-α/ß on pTreg differentiation and induction of peripheral tolerance, which may have important implications in the therapeutic control of inflammatory disorders, such as of autoimmune diseases.

Corrigendum to "Multicentre Harmonisation of a Six-Colour Flow Cytometry Panel for Naïve/Memory T Cell Immunomonitoring".

[This corrects the article DOI: 10.1155/2020/1938704.].

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.

Multicentre Harmonisation of a Six-Colour Flow Cytometry Panel for Naïve/Memory T Cell Immunomonitoring.

BACKGROUND: Personalised medicine in oncology needs standardised immunological assays. Flow cytometry (FCM) methods represent an essential tool for immunomonitoring, and their harmonisation is crucial to obtain comparable data in multicentre clinical trials. The objective of this study was to design a harmonisation workflow able to address the most effective issues contributing to intra- and interoperator variabilities in a multicentre project. METHODS: The Italian National Institute of Health (Istituto Superiore di Sanità, ISS) managed a multiparametric flow cytometric panel harmonisation among thirteen operators belonging to five clinical and research centres of Lazio region (Italy). The panel was based on a backbone mixture of dried antibodies (anti-CD3, anti-CD4, anti-CD8, anti-CD45RA, and anti-CCR7) to detect naïve/memory T cells, recognised as potential prognostic/predictive immunological biomarkers in cancer immunotherapies. The coordinating centre distributed frozen peripheral blood mononuclear cells (PBMCs) and fresh whole blood (WB) samples from healthy donors, reagents, and Standard Operating Procedures (SOPs) to participants who performed experiments by their own equipment, in order to mimic a real-life scenario. Operators returned raw and locally analysed data to ISS for central analysis and statistical elaboration. RESULTS: Harmonised and reproducible results were obtained by sharing experimental set-up and procedures along with centralising data analysis, leading to a reduction of cross-centre variability for naïve/memory subset frequencies particularly in the whole blood setting. CONCLUSION: Our experimental and analytical working process proved to be suitable for the harmonisation of FCM assays in a multicentre setting, where high-quality data are required to evaluate potential immunological markers, which may contribute to select better therapeutic options.

Clinical and Immunological Outcomes in High-Risk Resected Melanoma Patients Receiving Peptide-Based Vaccination and Interferon Alpha, With or Without Dacarbazine Preconditioning: A Phase II Study.

Clinical studies based on novel rationales and mechanisms of action of chemotherapy agents and cytokines can contribute to the development of new concepts and strategies of antitumor combination therapies. In previous studies, we investigated the paradoxical immunostimulating effects of some chemotherapeutics and the immunoadjuvant activity of interferon alpha (IFN-α) in preclinical and clinical models, thus unraveling novel rationales and mechanisms of action of chemotherapy agents and cytokines for cancer immunotherapy. Here, we carried out a randomized, phase II clinical trial, in which we analyzed the relapse-free (RFS) and overall survival (OS) of 34 completely resected stage III-IV melanoma patients, treated with peptide-based vaccination (Melan-A/MART-1 and NY-ESO-1) in combination with IFN-α2b, with (arm 2) or without (arm 1) dacarbazine preconditioning. All patients were included in the intention-to-treat analysis. At a median follow-up of 4.5 years (interquartile range, 15.4-81.0 months), the rates of RFS were 52.9 and 35.3% in arms 1 and 2, respectively. The 4.5-year OS rates were 68.8% in arm 1 and 62.7% in arm 2. No significant differences were observed between the two arms for both RFS and OS. Interestingly, the RFS and OS curves remained stable starting from 18 and 42 months, respectively. Grade 3 adverse events occurred in 5.9% of patients, whereas grade 4 events were not observed. Both treatments induced a significant expansion of vaccine-specific CD8+ T cells, with no correlation with the clinical outcome. However, treatment-induced increase of polyfunctionality and of interleukin 2 production by Melan-A-specific CD8+ T cells and expansion/activation of natural killer cells correlated with RFS, being observed only in nonrelapsing patients. Despite the recent availability of different therapeutic options, low-cost, low-toxic therapies with long-lasting clinical effects are still needed in patients with high-risk resected stage III/IV melanoma. The combination of peptide vaccination with IFN-α2b showed a minimal toxicity profile and resulted in encouraging RFS and OS rates, justifying further evaluation in clinical trials, which may include the use of checkpoint inhibitors to further expand the antitumor immune response and the clinical outcome. Clinical Trial Registration: https://www.clinicaltrialsregister.eu/ctr-search/search, identifier: 2008-008211-26.

Type I Interferons and Cancer: An Evolving Story Demanding Novel Clinical Applications.

The first report on the antitumor effects of interferon α/ß (IFN-I) in mice was published 50 years ago. IFN-α were the first immunotherapeutic drugs approved by the FDA for clinical use in cancer. However, their clinical use occurred at a time when most of their mechanisms of action were still unknown. These cytokines were being used as either conventional cytostatic drugs or non-specific biological response modifiers. Specific biological activities subsequently ascribed to IFN-I were poorly considered for their clinical use. Notably, a lot of the data in humans and mice underlines the importance of endogenous IFN-I, produced by both immune and tumor cells, in the control of tumor growth and in the response to antitumor therapies. While many oncologists consider IFN-I as "dead drugs", recent studies reveal new mechanisms of action with potential implications in cancer control and immunotherapy response or resistance, suggesting novel rationales for their usage in target and personalized anti-cancer treatments. In this Perspectives Article, we focus on the following aspects: (1) the added value of IFN-I for enhancing the antitumor impact of standard anticancer treatments (chemotherapy and radiotherapy) and new therapeutic approaches, such as check point inhibitors and epigenetic drugs; (2) the role of IFN-I in the control of cancer stem cells growth and its possible implications for the development of novel antitumor therapies; and (3) the role of IFN-I in the development of cancer vaccines and the intriguing therapeutic possibilities offered by in situ delivery of ex vivo IFN-stimulated dendritic cells.

Lenalidomide improves the therapeutic effect of an interferon-α-dendritic cell-based lymphoma vaccine.

The perspective of combining cancer vaccines with immunomodulatory drugs is currently regarded as a highly promising approach for boosting tumor-specific T cell immunity and eradicating residual malignant cells. The efficacy of dendritic cell (DC) vaccination in combination with lenalidomide, an anticancer drug effective in several hematologic malignancies, was investigated in a follicular lymphoma (FL) model. First, we evaluated the in vitro activity of lenalidomide in modulating the immune responses of lymphocytes co-cultured with a new DC subset differentiated with IFN-α (IFN-DC) and loaded with apoptotic lymphoma cells. We next evaluated the efficacy of lenalidomide and IFN-DC-based vaccination, either alone or in combination, in hu-PBL-NOD/SCID mice bearing established human lymphoma. We found that lenalidomide reduced Treg frequency and IL-10 production in vitro, improved the formation of immune synapses of CD8 + lymphocytes with lymphoma cells and enhanced anti-lymphoma cytotoxicity. Treatment of lymphoma-bearing mice with either IFN-DC vaccination or lenalidomide led to a significant decrease in tumor growth and lymphoma cell spread. Lenalidomide treatment was shown to substantially inhibit tumor-induced neo-angiogenesis rather than to exert a direct cytotoxic effect on lymphoma cells. Notably, the combined treatment with the vaccine plus lenalidomide was more effective than either single treatment, resulting in the significant regression of established tumors and delayed tumor regrowth upon treatment discontinuation. In conclusion, our data demonstrate that IFN-DC-based vaccination plus lenalidomide exert an additive therapeutic effect in xenochimeric mice bearing established lymphoma. These results may pave the way to evaluate this combination in the clinical ground.

In situ Vaccination by Direct Dendritic Cell Inoculation: The Coming of Age of an Old Idea?

For more than 25 years, dendritic cell (DC) based vaccination has flashily held promises to represent a therapeutic approach for cancer treatment. While the vast majority of studies has focused on the use of antigen loaded DC, the intratumoral delivery of unloaded DC aiming at in situ vaccination has gained much less attention. Such approach grounds on the ability of inoculated DC to internalize and process antigens directly released by tumor (usually in combination with cell-death-inducing agents) to activate broad patient-specific antitumor T cell response. In this review, we highlight the recent studies in both solid and hematological tumors showing promising clinical results and discuss the main pitfalls and advantages of this approach for endogenous cancer vaccination. Lastly, we discuss how in situ vaccination by DC inoculation may fit with current immunotherapy approaches to expand and prolong patient response.

Immune Dysfunctions and Immunotherapy in Colorectal Cancer: The Role of Dendritic Cells.

Colorectal cancer (CRC), a multi-step malignancy showing increasing incidence in today's societies, represents an important worldwide health issue. Exogenous factors, such as lifestyle, diet, nutrition, environment and microbiota, contribute to CRC pathogenesis, also influencing non neoplastic cells, including immune cells. Several immune dysfunctions were described in CRC patients at different disease stages. Many studies underline the role of microbiota, obesity-related inflammation, diet and host reactive cells, including dendritic cells (DC), in CRC pathogenesis. Here, we focused on DC, the main cells linking innate and adaptive anti-cancer immunity. Variations in the number and phenotype of circulating and tumor-infiltrating DC have been found in CRC patients and correlated with disease stages and progression. A critical review of DC-based clinical studies and of recent advances in cancer immunotherapy leads to consider new strategies for combining DC vaccination strategies with check-point inhibitors, thus opening perspectives for a more effective management of this neoplastic disease.

Clinical and Antitumor Immune Responses in Relapsed/Refractory Follicular Lymphoma Patients after Intranodal Injections of IFNα-Dendritic Cells and Rituximab: a Phase I Clinical Trial.

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.

Disruption of IFN-I Signaling Promotes HER2/Neu Tumor Progression and Breast Cancer Stem Cells.

Type I interferon (IFN-I) is a class of antiviral immunomodulatory cytokines involved in many stages of tumor initiation and progression. IFN-I acts directly on tumor cells to inhibit cell growth and indirectly by activating immune cells to mount antitumor responses. To understand the role of endogenous IFN-I in spontaneous, oncogene-driven carcinogenesis, we characterized tumors arising in HER2/neu transgenic (neuT) mice carrying a nonfunctional mutation in the IFNI receptor (IFNAR1). Such mice are unresponsive to this family of cytokines. Compared with parental neu+/- mice (neuT mice), IFNAR1-/- neu+/- mice (IFNAR-neuT mice) showed earlier onset and increased tumor multiplicity with marked vascularization. IFNAR-neuT tumors exhibited deregulation of genes having adverse prognostic value in breast cancer patients, including the breast cancer stem cell (BCSC) marker aldehyde dehydrogenase-1A1 (ALDH1A1). An increased number of BCSCs were observed in IFNAR-neuT tumors, as assessed by ALDH1A1 enzymatic activity, clonogenic assay, and tumorigenic capacity. In vitro exposure of neuT+ mammospheres and cell lines to antibodies to IFN-I resulted in increased frequency of ALDH+ cells, suggesting that IFN-I controls stemness in tumor cells. Altogether, these results reveal a role of IFN-I in neuT-driven spontaneous carcinogenesis through intrinsic control of BCSCs. Cancer Immunol Res; 6(6); 658-70. ©2018 AACR.

Role of interferon regulatory factor 1 in governing Treg depletion, Th1 polarization, inflammasome activation and antitumor efficacy of cyclophosphamide.

The antitumor effectiveness of cyclophosphamide (CTX) and other chemotherapeutics was shown to rely not only on direct cytotoxicity but also on immunogenic tumor cell death and systemic immunomodulatory mechanisms, including regulatory T cell (Treg) depletion, Th1 cell polarization, type I interferon (IFN) and proinflammatory cytokine production. IFN regulatory factor (IRF)-1 is a transcriptional regulator of IFNs and IFN-inducible genes, involved in the control of Th1 and Treg differentiation and in sterile inflammation. Aim of this study was to explore the role of IRF-1 in CTX-induced antitumor effects and related immune activities. This study shows for the first time that IRF-1 is important for the antitumor efficacy of CTX in mice. Moreover, experiments in tumor-bearing C57BL/6 mice showed that Irf1 gene expression in the spleen was transiently increased following CTX administration and correlated with the induction of Th1 cell expansion and of Il12p40 gene expression, which is the main Th1-driving cytokine. At the same time, CTX administration reduced both Foxp3 expression and Treg cell percentages. These effects were abrogated in Irf1-/- mice. Further experiments showed that the gene and/or protein expression of caspase-1, iNOS, IL-1ß, IL-6 and CXCL10 and the levels of nitric oxide were modulated following CTX in an IRF-1-direct- or -indirect-dependent manner, and highlighted the importance of caspase-1 in driving the sterile inflammatory response to CTX. Our data identify IRF-1 as important for the antitumor efficacy of CTX and for the regulation of many immunomodulatory activities of CTX, such as Th1 polarization, Treg depletion and inflammation.

Antitumor Effects of Epidrug/IFNα Combination Driven by Modulated Gene Signatures in Both Colorectal Cancer and Dendritic Cells.

Colorectal cancer results from the progressive accumulation of genetic and epigenetic alterations. IFN signaling defects play an important role in the carcinogenesis process, in which the inability of IFN transcription regulatory factors (IRF) to access regulatory sequences in IFN-stimulated genes (ISG) in tumors and in immune cells may be pivotal. We reported that low-dose combination of two FDA-approved epidrugs, azacytidine (A) and romidepsin (R), with IFNα2 (ARI) hampers the aggressiveness of both colorectal cancer metastatic and stem cells in vivo and triggers immunogenic cell death signals that stimulate dendritic cell (DC) function. Here, we investigated the molecular signals induced by ARI treatment and found that this drug combination increased the accessibility to regulatory sequences of ISGs and IRFs that were epigenetically silenced in both colorectal cancer cells and DCs. Likewise, specific ARI-induced histone methylation and acetylation changes marked epigenetically affected ISG promoters in both metastatic cancer cells and DCs. Analysis by ChIP-seq confirmed such ARI-induced epigenetically regulated IFN signature. The activation of this signal endowed DCs with a marked migratory capability. Our results establish a direct correlation between reexpression of silenced ISGs by epigenetic control and ARI anticancer activity and provide new knowledge for the development of innovative combined therapeutic strategies for colorectal cancer. Cancer Immunol Res; 5(7); 604-16. ©2017 AACR.

Chemo-immunotherapy induces tumor regression in a mouse model of spontaneous mammary carcinogenesis.

Tumor-specific immune tolerance represents an obstacle for the development of effective anti-tumor immune responses through cancer vaccines. We here evaluated the efficacy of chemo-immunotherapy in breaking tumor-specific immune tolerance in an almost incurable mouse model of spontaneous carcinogenesis.Transgenic HER-2/neu mice bearing large mammary tumors received the adoptive transfer of splenocytes and serum isolated from immune donors, with or without pre-conditioning with cyclophosphamide. Treatment efficacy was assessed by monitoring tumor growth by manual inspection and by magnetic resonance imaging. The same chemo-immunotherapy protocol was tested on tumor-free HER-2/neu mice, to evaluate the effects on tumor emergence.Our data show that chemo-immunotherapy hampered carcinogenesis and caused the regression of large mammary tumor lesions in tumor-bearing HER-2/neu mice. The complete eradication of a significant number of tumor lesions occurred only in mice receiving cyclophosphamide shortly before immunotherapy, and was associated with increased serum anti HER-2/p185 antibodies and tumor leukocyte infiltration. The same protocol significantly delayed the appearance of mammary tumors when administered to tumor-free HER-2/neu mice, indicating that this chemo-immunotherapy approach acted through the elicitation of an effective anti-tumor immune response. Overall, our data support the immune-modulatory role of chemotherapy in overcoming cancer immune tolerance when administered at lymphodepleting non-myeloablative doses shortly before transfer of antigen-specific immune cells and immunoglobulins. These findings open new perspectives on combining immune-modulatory chemotherapy and immunotherapy to overcome immune tolerance in cancer patients.

NK Cell Activation in the Antitumor Response Induced by IFN-α Dendritic Cells Loaded with Apoptotic Cells from Follicular Lymphoma Patients.

Follicular lymphoma (FL) is the most common form of indolent non-Hodgkin lymphoma. This malignancy is considered virtually incurable, with high response rates to therapy but frequent relapses. We investigated the ability of monocyte-derived dendritic cells generated in the presence of IFN-α and GM-CSF (IFN-DC) and loaded with apoptotic lymphoma cells to activate immune responses against FL cells, with the ultimate goal of designing novel patient-specific vaccination strategies for the treatment of FL. In this article, we show that apoptotic tumor cell-loaded IFN-DC from FL patients, which were cultured for 2 wk with autologous lymphocytes, led to Th1 response skewing, based on significantly higher levels of IFN-γ production and a remarkable increase in CD8(+) and NK cell frequency, consistent with the detection of enhanced cytotoxic effector function toward autologous FL cells. IFN-DC were found to promote efficient NK cell activation, increased expression of cytotoxicity receptors, and extensive IFN-γ production in the virtual absence of IL-10. Moreover, direct recognition and killing of primary autologous lymphoma cells by activated NK cells from FL patients was also demonstrated. A critical role was demonstrated for MHC class I-related chain A and B and membrane-bound IL-15 in IFN-DC-mediated NK cell activation and early IFN-γ production. The overall results indicate that IFN-DC loaded with autologous apoptotic FL cells represent a valuable tool for improving the potency of therapeutic cancer vaccines through the efficient induction of NK cell activation and promotion of CD8(+) T cell antitumor immunity.