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.

Intratumoral injection of IFN-alpha dendritic cells after dacarbazine activates anti-tumor immunity: results from a phase I trial in advanced melanoma.

BACKGROUND: Advanced melanoma patients have an extremely poor long term prognosis and are in strong need of new therapies. The recently developed targeted therapies have resulted in a marked antitumor effect, but most responses are partial and some degree of toxicity remain the major concerns. Dendritic cells play a key role in the activation of the immune system and have been typically used as ex vivo antigen-loaded cell drugs for cancer immunotherapy. Another approach consists in intratumoral injection of unloaded DCs that can exploit the uptake of a wider array of tumor-specific and individual unique antigens. However, intratumoral immunization requires DCs endowed at the same time with properties typically belonging to both immature and mature DCs (i.e. antigen uptake and T cell priming). DCs generated in presence of interferon-alpha (IFN-DCs), due to their features of partially mature DCs, capable of efficiently up-taking, processing and cross-presenting antigens to T cells, could successfully carry out this task. Combining intratumoral immunization with tumor-destructing therapies can induce antigen release in situ, facilitating the injected DCs in triggering an antitumor immune response. METHODS: We tested in a phase I clinical study in advanced melanoma a chemo-immunotherapy approach based on unloaded IFN-DCs injected intratumorally one day after administration of dacarbazine. Primary endpoint of the study was treatment safety and tolerability. Secondary endpoints were immune and clinical responses of patients. RESULTS: Six patients were enrolled, and only three completed the treatment. The chemo-immunotherapy was well tolerated with no major side effects. Three patients showed temporary disease stabilization and two of them showed induction of T cells specific for tyrosinase, NY-ESO-1 and gp100. Of interest, one patient showing a remarkable long-term disease stabilization kept showing presence of tyrosinase specific T cells in PBMC and high infiltration of memory T cells in the tumor lesion at 21 months. CONCLUSION: We tested a chemo-immunotherapeutic approach based on IFN-DCs injected intratumorally one day after DTIC in advanced melanoma. The treatment was well tolerated, and clinical and immunological responses, including development of vitiligo, were observed, therefore warranting additional clinical studies aimed at evaluating efficacy of this approach. TRIAL REGISTRATION: Trial Registration Number not publicly available due to EudraCT regulations: https://www.clinicaltrialsregister.eu/doc/EU_CTR_FAQ.pdf.

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.

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.

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.

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.

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.].

IL-33 Promotes CD11b/CD18-Mediated Adhesion of Eosinophils to Cancer Cells and Synapse-Polarized Degranulation Leading to Tumor Cell Killing.

Eosinophils are major effectors of Th2-related pathologies, frequently found infiltrating several human cancers. We recently showed that eosinophils play an essential role in anti-tumor responses mediated by immunotherapy with the 'alarmin' intereukin-33 (IL-33) in melanoma mouse models. Here, we analyzed the mechanisms by which IL-33 mediates tumor infiltration and antitumor activities of eosinophils. We show that IL-33 recruits eosinophils indirectly, via stimulation of tumor cell-derived chemokines, while it activates eosinophils directly, up-regulating CD69, the adhesion molecules ICAM-1 and CD11b/CD18, and the degranulation marker CD63. In co-culture experiments with four different tumor cell lines, IL-33-activated eosinophils established large numbers of stable cell conjugates with target tumor cells, with the polarization of eosinophil effector proteins (ECP, EPX, and granzyme-B) and CD11b/CD18 to immune synapses, resulting in efficient contact-dependent degranulation and tumor cell killing. In tumor-bearing mice, IL-33 induced substantial accumulation of degranulating eosinophils within tumor necrotic areas, indicating cytotoxic activity in vivo. Blocking of CD11b/CD18 signaling significantly reduced IL-33-activated eosinophils' binding and subsequent killing of tumor cells, indicating a crucial role for this integrin in triggering degranulation. Our findings provide novel mechanistic insights for eosinophil-mediated anti-tumoral function driven by IL-33. Treatments enabling tumor infiltration and proper activation of eosinophils may improve therapeutic response in cancer patients.

The Pleiotropic Immunomodulatory Functions of IL-33 and Its Implications in Tumor Immunity.

Interleukin-33 (IL-33) is a IL-1 family member of cytokines exerting pleiotropic activities. In the steady-state, IL-33 is expressed in the nucleus of epithelial, endothelial, and fibroblast-like cells acting as a nuclear protein. In response to tissue damage, infections or necrosis IL-33 is released in the extracellular space, where it functions as an alarmin for the immune system. Its specific receptor ST2 is expressed by a variety of immune cell types, resulting in the stimulation of a wide range of immune reactions. Recent evidences suggest that different IL-33 isoforms exist, in virtue of proteolytic cleavage or alternative mRNA splicing, with potentially different biological activity and functions. Although initially studied in the context of allergy, infection, and inflammation, over the past decade IL-33 has gained much attention in cancer immunology. Increasing evidences indicate that IL-33 may have opposing functions, promoting, or dampening tumor immunity, depending on the tumor type, site of expression, and local concentration. In this review we will cover the biological functions of IL-33 on various immune cell subsets (e.g., T cells, NK, Treg cells, ILC2, eosinophils, neutrophils, basophils, mast cells, DCs, and macrophages) that affect anti-tumor immune responses in experimental and clinical cancers. We will also discuss the possible implications of diverse IL-33 mutations and isoforms in the anti-tumor activity of the cytokine and as possible clinical biomarkers.

Gut Mesenchymal Stromal Cells in Immunity.

Mesenchymal stromal cells (MSCs), first found in bone marrow (BM), are the structural architects of all organs, participating in most biological functions. MSCs possess tissue-specific signatures that allow their discrimination according to their origin and location. Among their multiple functions, MSCs closely interact with immune cells, orchestrating their activity to maintain overall homeostasis. The phenotype of tissue MSCs residing in the bowel overlaps with myofibroblasts, lining the bottom walls of intestinal crypts (pericryptal) or interspersed within intestinal submucosa (intercryptal). In Crohn's disease, intestinal MSCs are tightly stacked in a chronic inflammatory milieu, which causes their enforced expression of Class II major histocompatibility complex (MHC). The absence of Class II MHC is a hallmark for immune-modulator and tolerogenic properties of normal MSCs and, vice versa, the expression of HLA-DR is peculiar to antigen presenting cells, that is, immune-activator cells. Interferon gamma (IFNγ) is responsible for induction of Class II MHC expression on intestinal MSCs. The reversal of myofibroblasts/MSCs from an immune-modulator to an activator phenotype in Crohn's disease results in the formation of a fibrotic tube subverting the intestinal structure. Epithelial metaplastic areas in this context can progress to dysplasia and cancer.

IL-33 restricts tumor growth and inhibits pulmonary metastasis in melanoma-bearing mice through eosinophils.

The alarmin IL-33 is an IL-1 family member that stimulates pleiotropic immune reactions depending on the target tissue and microenvironmental factors. In this study, we have investigated the role of IL-33/ST2 axis in antitumor response to melanoma. Injection of IL-33 in mice-bearing subcutaneous B16.F10 melanoma resulted in significant tumor growth delay. This effect was associated with intratumoral accumulation of CD8+ T cells and eosinophils, decrease of immunosuppressive myeloid cells, and a mixed Th1/Th2 cytokine expression pattern with local and systemic activation of CD8+ T and NK cells. Moreover, intranasal administration of IL-33 determined ST2-dependent eosinophil recruitment in the lung that prevented the onset of pulmonary metastasis after intravenous injection of melanoma cells. Accordingly, ST2-deficient mice developed pulmonary metastasis at higher extent than wild-type counterparts, associated with lower eosinophil frequencies in the lung. Of note, depletion of eosinophils by in vivo treatment with anti-Siglec-F antibody abolished the ability of IL-33 to both restrict primary tumor growth and metastasis formation. Finally, we show that IL-33 is able to activate eosinophils resulting in efficient killing of target melanoma cells, suggesting a direct antitumor activity of eosinophils following IL-33 treatment. Our results advocate for an eosinophil-mediated antitumoral function of IL-33 against melanoma, thus opening perspectives for novel cancer immunotherapy strategies.

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.

Cyclophosphamide induces a type I interferon-associated sterile inflammatory response signature in cancer patients' blood cells: implications for cancer chemoimmunotherapy.

PURPOSE: Certain chemotherapeutics, particularly cyclophosphamide, can enhance the antitumor efficacy of immunotherapy. A better understanding of the cellular and molecular basis of cyclophosphamide-mediated immunomodulation is needed to improve the efficacy of chemoimmunotherapy. EXPERIMENTAL DESIGN: Transcript profiling and flow cytometry were used to explore cyclophosphamide-induced immunoadjuvanticity in patients with hematologic malignancies. RESULTS: A single high-dose treatment rapidly (1-2 days) induced peripheral blood mononuclear cell (PBMC) transcriptional modulation, leading to reduction of cell-cycle and biosynthetic/metabolic processes and augmentation of DNA damage and cell death pathways (p53 signaling pathway), death-related scavenger receptors, antigen processing/presentation mediators, T-cell activation markers and, noticeably, a type I IFN (IFN-I) signature (OAS1, CXCL10, BAFF, IFITM2, IFI6, IRF5, IRF7, STAT2, UBE2L6, UNC93B1, ISG20L1, TYK2). Moreover, IFN-I-induced proinflammatory mediators (CXCL10, CCL2, IL-8, and BAFF) were increased in patients' plasma. Accordingly, cyclophosphamide induced the expansion/activation of CD14(+)CD16(+) monocytes, of HLA-DR(+), IL-8RA(+), and MARCO(+) monocytes/dendritic cells, and of CD69(+), OX40(+), and IL-8RA(+) lymphocytes. CONCLUSIONS: Altogether, these data identify the cyclophosphamide-induced immunomodulatory factors in humans and indicate that preconditioning chemotherapy may stimulate immunity as a consequence of danger perception associated with blood cell death, through p53 and IFN-I-related mechanisms.