Neutralization of NET-associated human ARG1 enhances cancer immunotherapy.

Myeloid cells can restrain antitumor immunity by metabolic pathways, such as the degradation of l-arginine, whose concentrations are regulated by the arginase 1 (ARG1) enzyme. Results from preclinical studies indicate the important role of arginine metabolism in pancreatic ductal adenocarcinoma (PDAC) progression, suggesting a potential for clinical application; however, divergent evolution in ARG1 expression and function in rodents and humans has restricted clinical translation. To overcome this dichotomy, here, we show that neutrophil extracellular traps (NETs), released by spontaneously activated neutrophils isolated from patients with PDAC, create a microdomain where cathepsin S (CTSS) cleaves human (h)ARG1 into different molecular forms endowed with enhanced enzymatic activity at physiological pH. NET-associated hARG1 suppresses T lymphocytes whose proliferation is restored by either adding a hARG1-specific monoclonal antibody (mAb) or preventing CTSS-mediated cleavage, whereas small-molecule inhibitors are not effective. We show that ARG1 blockade, combined with immune checkpoint inhibitors, can restore CD8+ T cell function in ex vivo PDAC tumors. Furthermore, anti-hARG1 mAbs increase the frequency of adoptively transferred tumor-specific CD8+ T cells in tumor and enhance the effectiveness of immune checkpoint therapy in humanized mice. Thus, this study shows that extracellular ARG1, released by activated myeloid cells, localizes in NETs, where it interacts with CTSS that in turn cleaves ARG1, producing major molecular forms endowed with different enzymatic activity at physiological pH. Once exocytosed, ARG1 activity can be targeted by mAbs, which bear potential for clinical application for the treatment of PDAC and require further exploration.

Interrupting the nitrosative stress fuels tumor-specific cytotoxic T lymphocytes in pancreatic cancer.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest tumors owing to its robust desmoplasia, low immunogenicity, and recruitment of cancer-conditioned, immunoregulatory myeloid cells. These features strongly limit the success of immunotherapy as a single agent, thereby suggesting the need for the development of a multitargeted approach. The goal is to foster T lymphocyte infiltration within the tumor landscape and neutralize cancer-triggered immune suppression, to enhance the therapeutic effectiveness of immune-based treatments, such as anticancer adoptive cell therapy (ACT). METHODS: We examined the contribution of immunosuppressive myeloid cells expressing arginase 1 and nitric oxide synthase 2 in building up a reactive nitrogen species (RNS)-dependent chemical barrier and shaping the PDAC immune landscape. We examined the impact of pharmacological RNS interference on overcoming the recruitment and immunosuppressive activity of tumor-expanded myeloid cells, which render pancreatic cancers resistant to immunotherapy. RESULTS: PDAC progression is marked by a stepwise infiltration of myeloid cells, which enforces a highly immunosuppressive microenvironment through the uncontrolled metabolism of L-arginine by arginase 1 and inducible nitric oxide synthase activity, resulting in the production of large amounts of reactive oxygen and nitrogen species. The extensive accumulation of myeloid suppressing cells and nitrated tyrosines (nitrotyrosine, N-Ty) establishes an RNS-dependent chemical barrier that impairs tumor infiltration by T lymphocytes and restricts the efficacy of adoptive immunotherapy. A pharmacological treatment with AT38 ([3-(aminocarbonyl)furoxan-4-yl]methyl salicylate) reprograms the tumor microenvironment from protumoral to antitumoral, which supports T lymphocyte entrance within the tumor core and aids the efficacy of ACT with telomerase-specific cytotoxic T lymphocytes. CONCLUSIONS: Tumor microenvironment reprogramming by ablating aberrant RNS production bypasses the current limits of immunotherapy in PDAC by overcoming immune resistance.

The immune modulatory effects of umbilical cord-derived mesenchymal stromal cells in severe COVID-19 pneumonia.

Coronavirus disease 2019 (COVID-19) may result in a life-threatening condition due to a hyperactive immune reaction to severe acute respiratory syndrome-coronavirus-2 infection, for which no effective treatment is available. Based on the potent immunomodulatory properties of mesenchymal stromal cells (MSCs), a growing number of trials are ongoing. This prompted us to carry out a thorough immunological study in a patient treated with umbilical cord-derived MSCs and admitted to the Intensive Care Unit for COVID-19-related pneumonia. The exploratory analyses were assessed on both peripheral blood and bronchoalveolar fluid lavage samples at baseline and after cellular infusion by means of single-cell RNA sequencing, flow cytometry, ELISA, and functional assays. Remarkably, a normalization of circulating T lymphocytes count paralleled by a reduction of inflammatory myeloid cells, and a decrease in serum levels of pro-inflammatory cytokines, mostly of interleukin-6 and tumor necrosis factor-α, were observed. In addition, a drop of plasma levels of those chemokines essential for neutrophil recruitment became evident that paralleled the decrease of lung-infiltrating inflammatory neutrophils. Finally, circulating monocytes and low-density gradient neutrophils acquired immunosuppressive function. This scenario was accompanied by an amelioration of respiratory, renal, inflammatory, and pro-thrombotic indexes. Our results provide the first immunological data possibly related to the use of umbilical cord-derived MSCs in severe COVID-19 context.

Deciphering the state of immune silence in fatal COVID-19 patients.

Since the beginning of the SARS-CoV-2 pandemic, COVID-19 appeared as a unique disease with unconventional tissue and systemic immune features. Here we show a COVID-19 immune signature associated with severity by integrating single-cell RNA-seq analysis from blood samples and broncho-alveolar lavage fluids with clinical, immunological and functional ex vivo data. This signature is characterized by lung accumulation of naïve lymphoid cells associated with a systemic expansion and activation of myeloid cells. Myeloid-driven immune suppression is a hallmark of COVID-19 evolution, highlighting arginase-1 expression with immune regulatory features of monocytes. Monocyte-dependent and neutrophil-dependent immune suppression loss is associated with fatal clinical outcome in severe patients. Additionally, our analysis shows a lung CXCR6+ effector memory T cell subset is associated with better prognosis in patients with severe COVID-19. In summary, COVID-19-induced myeloid dysregulation and lymphoid impairment establish a condition of 'immune silence' in patients with critical COVID-19.

Baricitinib restrains the immune dysregulation in patients with severe COVID-19.

BACKGROUNDPatients with coronavirus disease 2019 (COVID-19) develop pneumonia generally associated with lymphopenia and a severe inflammatory response due to uncontrolled cytokine release. These mediators are transcriptionally regulated by the JAK/STAT signaling pathways, which can be disabled by small molecules.METHODSWe treated a group of patients (n = 20) with baricitinib according to an off-label use of the drug. The study was designed as an observational, longitudinal trial and approved by the local ethics committee. The patients were treated with 4 mg baricitinib twice daily for 2 days, followed by 4 mg per day for the remaining 7 days. Changes in the immune phenotype and expression of phosphorylated STAT3 (p-STAT3) in blood cells were evaluated and correlated with serum-derived cytokine levels and antibodies against severe acute respiratory syndrome-coronavirus 2 (anti-SARS-CoV-2). In a single treated patient, we also evaluated the alteration of myeloid cell functional activity.RESULTSWe provide evidence that patients treated with baricitinib had a marked reduction in serum levels of IL-6, IL-1ß, and TNF-α, a rapid recovery of circulating T and B cell frequencies, and increased antibody production against the SARS-CoV-2 spike protein, all of which were clinically associated with a reduction in the need for oxygen therapy and a progressive increase in the P/F (PaO2, oxygen partial pressure/FiO2, fraction of inspired oxygen) ratio.CONCLUSIONThese data suggest that baricitinib prevented the progression to a severe, extreme form of the viral disease by modulating the patients' immune landscape and that these changes were associated with a safer, more favorable clinical outcome for patients with COVID-19 pneumonia.TRIAL REGISTRATIONClinicalTrials.gov NCT04438629.FUNDINGThis work was supported by the Fondazione Cariverona (ENACT Project) and the Fondazione TIM.

Disabled Homolog 2 Controls Prometastatic Activity of Tumor-Associated Macrophages.

Tumor-associated macrophages (TAM) are regulators of extracellular matrix (ECM) remodeling and metastatic progression, the main cause of cancer-associated death. We found that disabled homolog 2 mitogen-responsive phosphoprotein (DAB2) is highly expressed in tumor-infiltrating TAMs and that its genetic ablation significantly impairs lung metastasis formation. DAB2-expressing TAMs, mainly localized along the tumor-invasive front, participate in integrin recycling, ECM remodeling, and directional migration in a tridimensional matrix. DAB2+ macrophages escort the invasive dissemination of cancer cells by a mechanosensing pathway requiring the transcription factor YAP. In human lobular breast and gastric carcinomas, DAB2+ TAMs correlated with a poor clinical outcome, identifying DAB2 as potential prognostic biomarker for stratification of patients with cancer. DAB2 is therefore central for the prometastatic activity of TAMs. SIGNIFICANCE: DAB2 expression in macrophages is essential for metastasis formation but not primary tumor growth. Mechanosensing cues, activating the complex YAP-TAZ, regulate DAB2 in macrophages, which in turn controls integrin recycling and ECM remodeling in 3-D tissue matrix. The presence of DAB2+ TAMs in patients with cancer correlates with worse prognosis.This article is highlighted in the In This Issue feature, p. 1611.

Organoid-Transplant Model Systems to Study the Effects of Obesity on the Pancreatic Carcinogenesis in vivo.

Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer-related mortality among adults in developed countries. The discovery of the most common genetic alterations as well as the development of organoid models of pancreatic cancer have provided insight into the fundamental pathways driving tumor progression from a normal cell to non-invasive precursor lesion and finally to widely metastatic disease, offering new opportunities for identifying the key driver of cancer evolution. Obesity is one of the most serious public health challenges of the 21st century. Several epidemiological studies have shown the positive association between obesity and cancer-related morbidity/mortality, as well as poorer prognosis and treatment outcome. Despite strong evidence indicates a link between obesity and cancer incidence, the molecular basis of the initiating events remains largely elusive. This is mainly due to the lack of an accurate and reliable model of pancreatic carcinogenesis that mimics human obesity-associated PDAC, making data interpretation difficult and often confusing. Here we propose a feasible and manageable organoid-based preclinical tool to study the effects of obesity on pancreatic carcinogenesis. Therefore, we tracked the effects of obesity on the natural evolution of PDAC in a genetically defined transplantable model of the syngeneic murine pancreatic preneoplastic lesion (mP) and tumor (mT) derived-organoids that recapitulates the progression of human disease from early preinvasive lesions to metastatic disease. Our results suggest that organoid-derived transplant in obese mice represents a suitable system to study early steps of pancreatic carcinogenesis and supports the hypothesis that inflammation induced by obesity stimulates tumor progression and metastatization during pancreatic carcinogenesis.

The Engagement Between MDSCs and Metastases: Partners in Crime.

Tumor metastases represent the major cause of cancer-related mortality, confirming the urgent need to identify key molecular pathways and cell-associated networks during the early phases of the metastatic process to develop new strategies to either prevent or control distal cancer spread. Several data revealed the ability of cancer cells to establish a favorable microenvironment, before their arrival in distant organs, by manipulating the cell composition and function of the new host tissue where cancer cells can survive and outgrow. This predetermined environment is termed "pre-metastatic niche" (pMN). pMN development requires that tumor-derived soluble factors, like cytokines, growth-factors and extracellular vesicles, genetically and epigenetically re-program not only resident cells (i.e., fibroblasts) but also non-resident cells such as bone marrow-derived cells. Indeed, by promoting an "emergency" myelopoiesis, cancer cells switch the steady state production of blood cells toward the generation of pro-tumor circulating myeloid cells defined as myeloid-derived suppressor cells (MDSCs) able to sustain tumor growth and dissemination. MDSCs are a heterogeneous subset of myeloid cells with immunosuppressive properties that sustain metastatic process. In this review, we discuss current understandings of how MDSCs shape and promote metastatic dissemination acting in each fundamental steps of cancer progression from primary tumor to metastatic disease.

Immunosuppression by monocytic myeloid-derived suppressor cells in patients with pancreatic ductal carcinoma is orchestrated by STAT3.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is a highly devastating disease with an overall 5-year survival rate of less than 8%. New evidence indicates that PDAC cells release pro-inflammatory metabolites that induce a marked alteration of normal hematopoiesis, favoring the expansion and accumulation of myeloid-derived suppressor cells (MDSCs). We report here that PDAC patients show increased levels of both circulating and tumor-infiltrating MDSC-like cells. METHODS: The frequency of MDSC subsets in the peripheral blood was determined by flow cytometry in three independent cohorts of PDAC patients (total analyzed patients, n = 117). Frequency of circulating MDSCs was correlated with overall survival of PDAC patients. We also analyzed the frequency of tumor-infiltrating MDSC and the immune landscape in fresh biopsies. Purified myeloid cell subsets were tested in vitro for their T-cell suppressive capacity. RESULTS: Correlation with clinical data revealed that MDSC frequency was significantly associated with a shorter patients' overall survival and metastatic disease. However, the immunosuppressive activity of purified MDSCs was detectable only in some patients and mainly limited to the monocytic subset. A transcriptome analysis of the immunosuppressive M-MDSCs highlighted a distinct gene signature in which STAT3 was crucial for monocyte re-programming. Suppressive M-MDSCs can be characterized as circulating STAT3/arginase1-expressing CD14+ cells. CONCLUSION: MDSC analysis aids in defining the immune landscape of PDAC patients for a more appropriate diagnosis, stratification and treatment.

The Endless Saga of Monocyte Diversity.

Cancer immunotherapy relies on either restoring or activating the function of adaptive immune cells, mainly CD8+ T lymphocytes. Despite impressive clinical success, cancer immunotherapy remains ineffective in many patients due to the establishment of tumor resistance, largely dependent on the nature of tumor microenvironment. There are several cellular and molecular mechanisms at play, and the goal is to identify those that are clinically significant. Among the hematopoietic-derived cells, monocytes are endowed with high plasticity, responsible for their pro- and anti-tumoral function. Indeed, monocytes are involved in several cancer-associated processes such as immune-tolerance, metastatic spread, neoangiogenesis, and chemotherapy resistance; on the other hand, by presenting cancer-associated antigens, they can also promote and sustain anti-tumoral T cell response. Recently, by high throughput technologies, new findings have revealed previously underappreciated, profound transcriptional, epigenetic, and metabolic differences among monocyte subsets, which complement and expand our knowledge on the monocyte ontogeny, recruitment during steady state, and emergency hematopoiesis, as seen in cancer. The subdivision into discrete monocytes subsets, both in mice and humans, appears an oversimplification, whereas continuum subsets development is best for depicting the real condition. In this review, we examine the evidences sustaining the existence of a monocyte heterogeneity along with functional activities, at the primary tumor and at the metastatic niche. In particular, we describe how tumor-derived soluble factors and cell-cell contact reprogram monocyte function. Finally, we point out the role of monocytes in preparing and shaping the metastatic niche and describe relevant targetable molecules altering monocyte activities. We think that exploiting monocyte complexity can help identifying key pathways important for the treatment of cancer and several conditions where these cells are involved.

Nicotinamide Phosphoribosyltransferase Acts as a Metabolic Gate for Mobilization of Myeloid-Derived Suppressor Cells.

Cancer induces alteration of hematopoiesis to fuel disease progression. We report that in tumor-bearing mice the macrophage colony-stimulating factor elevates the myeloid cell levels of nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in the NAD salvage pathway, which acts as negative regulator of the CXCR4 retention axis of hematopoietic cells in the bone marrow. NAMPT inhibits CXCR4 through a NAD/Sirtuin 1-mediated inactivation of HIF1α-driven CXCR4 gene transcription, leading to mobilization of immature myeloid-derived suppressor cells (MDSC) and enhancing their production of suppressive nitric oxide. Pharmacologic inhibition or myeloid-specific ablation of NAMPT prevented MDSC mobilization, reactivated specific antitumor immunity, and enhanced the antitumor activity of immune checkpoint inhibitors. Our findings identify NAMPT as a metabolic gate of MDSC precursor function, providing new opportunities to reverse tumor immunosuppression and to restore clinical efficacy of immunotherapy in patients with cancer. SIGNIFICANCE: These findings identify NAMPT as a metabolic gate of MDSC precursor function, providing new opportunities to reverse tumor immunosuppression and to restore clinical efficacy of immunotherapy in cancer patients.

Induction of immunosuppressive functions and NF-κB by FLIP in monocytes.

Immunosuppression is a hallmark of tumor progression, and treatments that inhibit or deplete monocytic myeloid-derived suppressive cells could promote anti-tumor immunity. c-FLIP is a central regulator of caspase-8-mediated apoptosis and necroptosis. Here we show that low-dose cytotoxic chemotherapy agents cause apoptosis linked to c-FLIP down-regulation selectively in monocytes. Enforced expression of c-FLIP or viral FLIP rescues monocytes from cytotoxicity and concurrently induces potent immunosuppressive activity, in T cell cultures and in vivo models of tumor progression and immunotherapy. FLIP-transduced human blood monocytes can suppress graft versus host disease. Neither expression of FLIP in granulocytes nor expression of other anti-apoptotic genes in monocytes conferred immunosuppression, suggesting that FLIP effects on immunosuppression are specific to monocytic lineage and distinct from death inhibition. Mechanistically, FLIP controls a broad transcriptional program, partially by NF-κB activation. Therefore, modulation of FLIP in monocytes offers a means to elicit or block immunosuppressive myeloid cells.

Hyperthermic treatment at 56 °C induces tumour-specific immune protection in a mouse model of prostate cancer in both prophylactic and therapeutic immunization regimens.

Most active cancer immunotherapies able to induce a long-lasting protection against tumours are based on the activation of tumour-specific cytotoxic T lymphocytes (CTLs). Cell death by hyperthermia induces apoptosis followed by secondary necrosis, with the production of factors named "danger associated molecular pattern" (DAMP) molecules (DAMPs), that activate dendritic cells (DCs) to perform antigen uptake, processing and presentation, followed by CTLs cross priming. In many published studies, hyperthermia treatment of tumour cells is performed at 42-45 °C; these temperatures mainly promote cell surface expression of DAMPs. Treatment at 56 °C of tumour cells was shown to induce DAMPs secretion rather than their cell surface expression, improving DC activation and CTL cross priming in vitro. Thus we tested the relevance of this finding in vivo on the generation of a tumour-specific memory immune response, in the TRAMP-C2 mouse prostate carcinoma transplantable model. TRAMP-C2 tumour cells treated at 56 °C were able not only to activate DCs in vitro but also to trigger a tumour-specific CTL-dependent immune response in vivo. Prophylactic vaccination with 56 °C-treated TRAMP-C2 tumour cells alone provided protection against TRAMP-C2 tumour growth in vivo, whilst in the therapeutic regimen, control of tumour growth was achieved combining immunization with adjuvant chemotherapy.

Effective control of acute myeloid leukaemia and acute lymphoblastic leukaemia progression by telomerase specific adoptive T-cell therapy.

Telomerase (TERT) is a ribonucleoprotein enzyme that preserves the molecular organization at the ends of eukaryotic chromosomes. Since TERT deregulation is a common step in leukaemia, treatments targeting telomerase might be useful for the therapy of hematologic malignancies. Despite a large spectrum of potential drugs, their bench-to-bedside translation is quite limited, with only a therapeutic vaccine in the clinic and a telomerase inhibitor at late stage of preclinical validation. We recently demonstrated that the adoptive transfer of T cell transduced with an HLA-A2-restricted T-cell receptor (TCR), which recognize human TERT with high avidity, controls human B-cell chronic lymphocytic leukaemia (B-CLL) progression without severe side-effects in humanized mice. In the present report, we show the ability of our approach to limit the progression of more aggressive leukemic pathologies, such as acute myeloid leukaemia (AML) and B-cell acute lymphoblastic leukaemia (B-ALL). Together, our findings demonstrate that TERT-based adoptive cell therapy is a concrete platform of T cell-mediated immunotherapy for leukaemia treatment.

Antitumor immunization of mothers delays tumor development in cancer-prone offspring.

Maternal immunization is successfully applied against some life-threatening infectious diseases as it can protect the mother and her offspring through the passive transfer of maternal antibodies. Here, we sought to evaluate whether the concept of maternal immunization could also be applied to cancer immune-prevention. We have previously shown that antibodies induced by DNA vaccination against rat Her2 (neu) protect heterozygous neu-transgenic female (BALB-neuT) mice from autochthonous mammary tumor development. We, herein, seek to evaluate whether a similar maternal immunization can confer antitumor protection to BALB-neuT offspring. Significantly extended tumor-free survival was observed in BALB-neuT offspring born and fed by mothers vaccinated against neu, as compared to controls. Maternally derived anti-neu immunoglobulin G (IgG) was successfully transferred from mothers to newborns and was responsible for the protective effect. Vaccinated mothers and offspring also developed active immunity against neu as revealed by the presence of T-cell-mediated cytotoxicity against the neu immunodominant peptide. This active response was due to the milk transfer of immune complexes that were formed between the neu extracellular domain, shed from vaccine-transfected muscle cells, and the anti-neu IgG induced by the vaccine. These findings show that maternal immunization has the potential to hamper mammary cancer in genetically predestinated offspring and to develop into applications against lethal neonatal cancer diseases for which therapeutic options are currently unavailable.