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The p140Cap adaptor protein is a tumor suppressor in breast cancer associated with a favorable prognosis. Here we highlight a function of p140Cap in orchestrating local and systemic tumor-extrinsic events that eventually result in inhibition of the polymorphonuclear myeloid-derived suppressor cell function in creating an immunosuppressive tumor-promoting environment in the primary tumor, and premetastatic niches at distant sites. Integrative transcriptomic and preclinical studies unravel that p140Cap controls an epistatic axis where, through the upstream inhibition of β-Catenin, it restricts tumorigenicity and self-renewal of tumor-initiating cells limiting the release of the inflammatory cytokine G-CSF, required for polymorphonuclear myeloid-derived suppressor cells to exert their local and systemic tumor conducive function. Mechanistically, p140Cap inhibition of β-Catenin depends on its ability to localize in and stabilize the β-Catenin destruction complex, promoting enhanced β-Catenin inactivation. Clinical studies in women show that low p140Cap expression correlates with reduced presence of tumor-infiltrating lymphocytes and more aggressive tumor types in a large cohort of real-life female breast cancer patients, highlighting the potential of p140Cap as a biomarker for therapeutic intervention targeting the β-Catenin/ Tumor-initiating cells /G-CSF/ polymorphonuclear myeloid-derived suppressor cell axis to restore an efficient anti-tumor immune response.

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.

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-1β, 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.

Arginine metabolism plays a significant role in regulating cell function, affecting tumor growth and metastatization. To study the effect of the arginine-catabolizing enzyme Arginase1 (ARG1) on tumor microenvironment, we generated a mouse model of mammary carcinogenesis by crossbreeding a transgenic mouse line overexpressing ARG1 in macrophages (FVBArg+/+) with the MMTV-Neu mouse line (FVBNeu+/+). This double transgenic line (FVBArg+/−;Neu+/+) showed a significant shortening in mammary tumor latency, and an increase in the number of mammary nodules. Transfer of tumor cells from FVBNeu+/+ into either FVB wild type or FVBArg+/+ mice resulted in increase regulatory T cells in the tumor infiltrate, suggestive of an impaired antitumor immune response. However, we also found increased frequency of tumor stem cells in tumors from FVBArg+/−;Neu+/+ transgenic compared with FVBNeu+/+ mice, suggesting that increased arginine metabolism in mammary tumor microenvironment may supports the cancer stem cells niche. We provide in vivo evidence of a novel, yet unexploited, mechanism through which ARG1 may contribute to tumor development.

Myeloid-derived suppressor cells (MDSC) include immature monocytic (M-MDSC) and granulocytic (PMN-MDSC) cells that share the ability to suppress adaptive immunity and to hinder the effectiveness of anticancer treatments. Of note, in response to IFNγ, M-MDSCs release the tumor-promoting and immunosuppressive molecule nitric oxide (NO), whereas macrophages largely express antitumor properties. Investigating these opposing activities, we found that tumor-derived prostaglandin E2 (PGE2) induces nuclear accumulation of p50 NF-κB in M-MDSCs, diverting their response to IFNγ toward NO-mediated immunosuppression and reducing TNFα expression. At the genome level, p50 NF-κB promoted binding of STAT1 to regulatory regions of selected IFNγ-dependent genes, including inducible nitric oxide synthase (Nos2). In agreement, ablation of p50 as well as pharmacologic inhibition of either the PGE2 receptor EP2 or NO production reprogrammed M-MDSCs toward a NOS2low/TNFαhigh phenotype, restoring the in vivo antitumor activity of IFNγ. Our results indicate that inhibition of the PGE2/p50/NO axis prevents MDSC-suppressive functions and restores the efficacy of anticancer immunotherapy. Significance: Tumor-derived PGE2-mediated induction of nuclear p50 NF-κB epigenetically reprograms the response of monocytic cells to IFNγ toward an immunosuppressive phenotype, thus retrieving the anticancer properties of IFNγ.

BackgroundPancreatic 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).MethodsWe 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.ResultsPDAC 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.ConclusionsTumor microenvironment reprogramming by ablating aberrant RNS production bypasses the current limits of immunotherapy in PDAC by overcoming immune resistance.

One of the mechanisms that characterizes the aging process of different organs is the accumulation of fat. Different authors have demonstrated that adipose tissue replaces the loss of other cell types, deriving from mesenchymal cells. During aging, there is substitution or trans-differentiation of mesenchymal cells with other cells having the same embryological origin. Newly formed adipocytes were also observed in the trabecular matrix of elderly people’s bones, associated with myeloid cells. In this study, we have investigated the relationship between immature myeloid-derived suppressor cells (I-MDSCs) and mesenchymal stem cells (MSCs) in bone marrow (BM) samples harvested from 57 patients subjected to different orthopedic surgeries. Patients aged from 18 to 92 years were considered in order to compare the cellular composition of bone marrow of young and elderly people, considered a biomarker of immunity, inflammation, and bone preservation. The I-MDSC percentage was stable during aging, but in elderly people, it was possible to observe a strong basal immunosuppression of autologous and heterologous T cells’ proliferation. We hypothesized that this pattern observed in elders depends on the progressive accumulation in the BM of activating stimuli, including cell–cell contact, or the production of different cytokines and proteins that induce the differentiation of bone marrow mesenchymal stem cells in adipocytes. The collected data provided underline the importance of specific biomarkers of aging that promote a reduction in immune response and incremented inflammatory pathways, leading to bone reabsorption in elderly people.

Vaccination has been a game changer in our efforts to address the coronavirus disease 2019 (COVID-19) pandemic. However, the disease might still represent a clinical crisis for several more years, in part because of the inevitable emergence of variants capable of evading the preexisting immunity. Drugs affecting viral spread will help curtail transmission, but therapeutics are needed to treat the more severe cases requiring hospitalization. A deep analysis of the evolving immune landscape of COVID-19 suggests that understanding the molecular bases of the distinct clinical stages is paramount if we are to limit the burden of inflammation, which can lead to death in frail individuals, according to age, sex, and comorbidities. Different phases can be defined using immune biomarkers and need specific therapeutic approaches, tailored to the underlying immune contexture.

Purpose of review This review aims to provide an updated report in regards to the correlation between vaccines and anaphylaxis and the related risk in the population. Recent findings Initial reports showed higher incidence of anaphylaxis following messenger RNA COVID-19 vaccines compared with 'routine' vaccinations, likely influenced by the great attention paid to these 'new' vaccines. However, anaphylaxis has still to be considered quite rare and its incidence will be systematically reconsidered in the light of additional data collected. Summary Adverse reactions to vaccines are commonly reported but most of them are nonspecific mild events, whereas vaccine-related anaphylaxis is considered a rare event, with an incidence rate equal to 1.3 cases per million vaccine doses administered. As anaphylaxis reports usually start to be reported to passive pharmacovigilance during postmarketing surveillance, the first data are used to be influenced by under- and over-reporting and lack of denominators and following studies are needed to confirm the causal relationship. This might create an initial overcautiously approach to new immunization practices but, being anaphylaxis a potential life-threatening event, every suspected contraindication has to be deepened to maximize effectiveness and safety profile and constantly redefined not to exclude an overestimated population group who could receive the vaccine uneventfully.

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.

Here we report the identification of human CD66b

Inflammatory responses rapidly detect pathogen invasion and mount a regulated reaction. However, dysregulated anti-pathogen immune responses can provoke life-threatening inflammatory pathologies collectively known as cytokine release syndrome (CRS), exemplified by key clinical phenotypes unearthed during the SARS-Cov-2 pandemic. The underlying pathophysiology of CRS remains elusive. We found that FLIP, a protein that controls caspase-8 death pathways, was highly expressed in myeloid cells of COVID-19 lungs. FLIP controlled CRS by fueling a STAT3-dependent inflammatory program. Indeed, constitutive expression of a viral FLIP homologue in myeloid cells triggered a STAT3-linked, progressive and fatal inflammatory syndrome in mice, characterized by elevated cytokine output, lymphopenia, lung injury and multiple organ dysfunctions that mimicked human CRS. As STAT3-targeting approaches relieved inflammation, immune disorders, and organ failures in these mice, targeted intervention towards this pathway could suppress the lethal CRS inflammatory state.One sentence summaryFLIP-expressing myeloid cells are key drivers of CRS through aberrant overexpression of STAT3 pathway. STAT3-targeting is effective in mitigating CRS like severe COVID-19.

Expression of the L-arginine catabolizing enzyme arginase 1 (ARG1) is a central immunosuppressive mechanism mediated by tumor-educated myeloid cells. Increased activity of ARG1 promotes the formation of an immunosuppressive microenvironment and leads to a more aggressive phenotype in many cancers. Intrinsic T-cell immunity against ARG1-derived epitopes in the peripheral blood of cancer patients and healthy subjects has previously been demonstrated. To evaluate the antitumor efficacy of ARG1-derived peptide vaccines as a monotherapy and as a combinational therapy with checkpoint blockade, different in vivo syngeneic mouse tumor models were utilized. To evaluate the antitumor effects, flow cytometry analysis and IHC were performed on tumors, and ELISPOT assays were performed to characterize immune responses. We show that ARG1-targeting therapeutic vaccines were able to activate endogenous antitumor immunity in several in vivo syngeneic mouse tumor models and to modulate the cell composition of the tumor microenvironment without causing any associated side effects or systemic toxicity. ARG1-targeting vaccines in combination with anti–PD-1 also resulted in increased T-cell infiltration, decreased ARG1 expression, reduced suppressive function of tumor-educated myeloid cells, and a shift in the M1/M2 ratio of tumor-infiltrating macrophages. These results indicated that the induced shift toward a more proinflammatory microenvironment by ARG1-targeting immunotherapy favors effective tumor control when combined with anti–PD-1 checkpoint blockade. Our data illustrate the ability of ARG1-based immune modulatory vaccination to elicit antigen-specific immunosurveillance and imply the feasibility of this novel immunotherapeutic approach for clinical translation.

The diversity of regulatory T (T(reg)) cells in health and in disease remains unclear. Individuals with colorectal cancer harbor a subpopulation of RORγt(+) T(reg) cells with elevated expression of β-catenin and pro-inflammatory properties. Here we show progressive expansion of RORγt(+) T(reg) cells in individuals with inflammatory bowel disease during inflammation and early dysplasia. Activating Wnt–β-catenin signaling in human and murine T(reg) cells was sufficient to recapitulate the disease-associated increase in the frequency of RORγt(+) T(reg) cells coexpressing multiple pro-inflammatory cytokines. Binding of the β-catenin interacting partner, TCF-1, to DNA overlapped with Foxp3 binding at enhancer sites of pro-inflammatory pathway genes. Sustained Wnt–β-catenin activation induced newly accessible chromatin sites in these genes and upregulated their expression. These findings indicate that TCF-1 and Foxp3 together limit the expression of pro-inflammatory genes in T(reg) cells. Activation of β-catenin signaling interferes with this function and promotes the disease-associated RORγt(+) T(reg) phenotype.

Immunotherapy has revolutionized cancer treatment over the past decade. Nonetheless, prolonged survival is limited to relatively few patients. Cancers enforce a multifaceted immune-suppressive network whose nature is progressively shaped by systemic and local cues during tumor development. Monocytes bridge innate and adaptive immune responses and can affect the tumor microenvironment through various mechanisms that induce immune tolerance, angiogenesis, and increased dissemination of tumor cells. Yet monocytes can also give rise to antitumor effectors and activate antigen-presenting cells. This yin-yang activity relies on the plasticity of monocytes in response to environmental stimuli. In this review, we summarize current knowledge of the ontogeny, heterogeneity, and functions of monocytes and monocyte-derived cells in cancer, pinpointing the main pathways that are important for modeling the immunosuppressive tumor microenvironment.

Current understanding of the complex pathogenesis of COVID-19 interstitial pneumonia pathogenesis in the light of biopsies carried out in early/moderate phase and histology data obtained at postmortem analysis is discussed. In autopsies the most observed pattern is diffuse alveolar damage with alveolar-epithelial type-II cell hyperplasia, hyaline membranes, and frequent thromboembolic disease. However, these observations cannot explain some clinical, radiological and physiopathological features observed in SARS-CoV-2 interstitial pneumonia, including the occurrence of vascular enlargement on CT and preserved lung compliance in subjects even presenting with or developing respiratory failure. Histological investigation on early-phase pneumonia on perioperative samples and lung biopsies revealed peculiar morphological and morpho-phenotypical changes including hyper-expression of phosphorylated STAT3 and immune checkpoint molecules (PD-L1 and IDO) in alveolar-epithelial and endothelial cells. These features might explain in part these discrepancies.

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., Integrated studies of matched tissue sites and cell types in COVID-19 patients are important to define the immune mechanisms of pathology. Here, the authors describe an immune signature in fatal COVID-19 patients harmonizing single-cell RNA sequencing of blood and matched BAL cells with deep clinical, immunological and functional data.

Local delivery of anticancer agents has the potential to maximize treatment efficacy and minimize the acute and long-term systemic toxicities. Here we used unsupervised systematic evolution of ligands by exponential enrichment (SELEX) to identify four RNA aptamers that specifically recognized mouse and human myeloid cells infiltrating tumors but not their peripheral or circulating counterparts in multiple mouse models and from patients with head and neck squamous cell carcinoma (HNSCC). The use of these aptamers conjugated to doxorubicin enhanced the accumulation and bystander release of the chemotherapeutic drug in both primary and metastatic tumor sites in breast and fibrosarcoma mouse models. In the 4T1 mammary carcinoma model, these doxorubicin-conjugated aptamers outperformed Doxil, the first clinically approved highly optimized nanoparticle for targeted chemotherapy, by promoting tumor regression with no detected toxicity measured as weight loss and by blood chemistry after just 3 administrations. These RNA aptamers recognized tumor infiltrating myeloid cells in a variety of mouse tumors in vivo and from human HNSCC ex vivo. This suggests their use for the detection of myeloid-derived suppressor cells (MDSCs) in human and for a targeted delivery of chemotherapy to the tumor microenvironment in multiple malignancies.

Coronavirus disease 2019 (SARS-CoV2) is an active global health threat for which treatments are desperately being sought. Even though most people infected experience mild to moderate respiratory symptoms and recover with supportive care, certain vulnerable hosts develop severe clinical deterioration. While several drugs are currently being investigated in clinical trials, there are currently no approved treatments or vaccines for COVID-19 and hence there is an unmet need to explore additional therapeutic options. At least three inflammatory disorders or syndromes associated with immune dysfunction have been described in the context of cellular therapy. Specifically, Cytokine Release Syndrome (CRS), Immune Reconstitution Inflammatory Syndrome (IRIS), and Secondary Hemophagocytic Lymphohistiocytosis (sHLH) all have clinical and laboratory characteristics in common with COVID19 and associated therapies that could be worth testing in the context of clinical trials. Here we discuss these diseases, their management, and potential applications of these treatment in the context of COVID-19. We also discuss current cellular therapies that are being evaluated for the treatment of COVID-19 and/or its associated symptoms.

Patients with ulcerative colitis (UC) have an increased risk of developing colitis-associated colon cancer (CACC). Changes in glycosylation of the oncoprotein MUC1 commonly occur in chronic inflammation, including UC, and this abnormally glycosylated MUC1 promotes cancer development and progression. It is not known what causes changes in glycosylation of MUC1. Gene expression profiling of myeloid cells in inflamed and malignant colon tissues showed increased expression levels of inflammatory macrophage-associated cytokines compared to normal tissues. We analyzed the involvement of macrophage-associated cytokines in the induction of aberrant MUC1 glycoforms. A co-culture system was used to examine the effects of M1 and M2 macrophages on glycosylation-related enzymes in colon cancer cells. M2-like macrophages induced the expression of the glycosyltransferase ST6GALNAC1, an enzyme that adds sialic acid to O-linked GalNAc residues, promoting the formation of tumor-associated sialyl-Tn (sTn) O-glycans. Immunostaining of UC and CACC tissue samples confirmed the elevated number of M2-like macrophages as well as high expression of ST6GALNAC1 and the altered MUC1-sTn glycoform on colon cells. Cytokine arrays and blocking antibody experiments indicated that the macrophage-dependent ST6GALNAC1 activation was mediated by IL-13 and CCL17. We demonstrated that IL-13 promoted phosphorylation of STAT6 to activate transcription of ST6GALNAC1. A computational model of signaling pathways was assembled and used to test IL-13 inhibition as a possible therapy. Our findings revealed a novel cellular cross-talk between colon cells and macrophages within the inflamed and malignant colon that contributes to the pathogenesis of UC and CACC.

Immunotherapy has emerged as a potent alternative for cancer treatment, unfortunately, the clinical benefit remains limited to few patients and immunotherapy resistance due to immunosuppressive tumor microenvironment represents the major reason of such a failure. Arginase-1 is one of the enzymes contributing to the establishment of such immunosuppression. Among the human immune cells, polymorphonuclear cells (PMNs) represent the major source of arginase-1, while myeloid-derived suppressor cells (MDSCs) are the main arginase-1 producing cells in mice. Due to arginase-1 potential impact in dampening the immune response, there is a growing interest in assaying arginase-1 levels and functions. Thus, in this chapter we propose how to evaluate the expression and activity of arginase in human peripheral blood-derived PMNs and in MDSCs isolated from tumor-bearing mice.

Supplemental Digital Content is available in the text., Objective: Pulmonary thrombosis is observed in severe acute respiratory syndrome coronavirus 2 pneumonia. Aim was to investigate whether subpopulations of platelets were programmed to procoagulant and inflammatory activities in coronavirus disease 2019 (COVID-19) patients with pneumonia, without comorbidities predisposing to thromboembolism. Approach and Results: Overall, 37 patients and 28 healthy subjects were studied. Platelet-leukocyte aggregates, platelet-derived microvesicles, the expression of P-selectin, and active fibrinogen receptor on platelets were quantified by flow cytometry. The profile of 45 cytokines, chemokines, and growth factors released by platelets was defined by immunoassay. The contribution of platelets to coagulation factor activity was selectively measured. Numerous platelet-monocyte (mean±SE, 67.9±4.9%, n=17 versus 19.4±3.0%, n=22; P

BackgroundMyeloid Derived Suppressor Cells (MDSCs) and Tumor-Associated Macrophages (TAMs) are two of the major players involved in the inhibition of anti-tumor immune response in cancer patients, leading to poor prognosis. Selective targeting of myeloid cells has therefore become an attractive therapeutic strategy to relieve immunosuppression and, in this frame, we previously demonstrated that lipid nanocapsules (LNCs) loaded with lauroyl-modified gemcitabine efficiently target monocytic MDSCs in melanoma patients. In this study, we investigated the impact of the physico-chemical characteristics of LNCs, namely size and surface potential, towards immunosuppressive cell targeting. We exploited myeloid cells isolated from glioblastoma patients, which play a relevant role in the immunosuppression, to demonstrate that tailored nanosystems can target not only tumor cells but also tumor-promoting cells, thus constituting an efficient system that could be used to inhibit their function.ResultsThe incorporation of different LNC formulations with a size of 100 nm, carrying overall positive, neutral or negative charge, was evaluated on leukocytes and tumor-infiltrating cells freshly isolated from glioblastoma patients. We observed that the maximum LNC uptake was obtained in monocytes with neutral 100 nm LNCs, while positively charged 100 nm LNCs were more effective on macrophages and tumor cells, maintaining at low level the incorporation by T cells. The mechanism of uptake was elucidated, demonstrating that LNCs are incorporated mainly by caveolae-mediated endocytosis. ConclusionsWe demonstrated that LNCs can be directed towards immunosuppressive cells by simply modulating their size and charge and provided a novel approach to exploit nanosystems for anticancer treatment in the frame of immunotherapy.

Some enzymes degrading amino acids have evolved in mammals to dampen immune responses and maintain peripheral tolerance. The enzymes metabolizing l-arginine and l-tryptophan are particularly powerful, contributing to restrain immunity towards fetal tissues and establish neonatal tolerance. Solid tumors can hijack these formidable pathways to construct a microenvironment highly unfavorable to anti-tumor T lymphocytes able to recognize them, one of mechanisms for their immune evasion. In this review, we analyze emerging concepts in the cross-talk between cells expressing these enzymes, their immune regulatory functions and pharmacological approaches that can target them to enhance cancer immunotherapy.

As cancer strikes, individuals vary not only in terms of factors that contribute to its occurrence and development, but as importantly, in their capacity to respond to treatment. While exciting new therapeutic options that mobilize the immune system against cancer have led to breakthroughs for a variety of malignancies, success is limited to a subset of patients. Pre-existing immunological features of both the host and the tumor may contribute to how patients will eventually fare with immunotherapy. A broad understanding of baseline immunity, both in the periphery and in the tumor microenvironment, is needed in order to fully realize the potential of cancer immunotherapy. Such interrogation of the tumor, blood, and host immune parameters prior to treatment is expected to identify biomarkers predictive of clinical outcome as well as to elucidate why some patients fail to respond to immunotherapy. To approach these opportunities for progress, the Society for Immunotherapy of Cancer (SITC) reconvened the Immune Biomarkers Task Force. Comprised of an international multidisciplinary panel of experts, Working Group 4 sought to make recommendations that focus on the complexity of the tumor microenvironment, with its diversity of immune genes, proteins, cells, and pathways naturally present at baseline and in circulation, and novel tools to aid in such broad analyses.

General control nonderepressible 2 (GCN2) is an environmental sensor controlling transcription and translation in response to nutrient availability. Although GCN2 is a putative therapeutic target for immuno-oncology, its role in shaping the immune response to tumors is poorly understood. Here, we used mass cytometry, transcriptomics, and transcription factor-binding analysis to determine the functional impact of GCN2 on the myeloid phenotype and immune responses in melanoma. We found that myeloid-lineage deletion of GCN2 drives a shift in the phenotype of tumor-associated macrophages and myeloid-derived suppressor cells (MDSCs) that promotes antitumor immunity. Time-of-flight mass cytometry (CyTOF) and single-cell RNA sequencing showed that this was due to changes in the immune microenvironment with increased proinflammatory activation of macrophages and MDSCs and interferon-γ expression in intratumoral CD8+ T cells. Mechanistically, GCN2 altered myeloid function by promoting increased translation of the transcription factor CREB-2/ATF4, which was required for maturation and polarization of macrophages and MDSCs in both mice and humans, whereas targeting Atf4 by small interfering RNA knockdown reduced tumor growth. Last, analysis of patients with cutaneous melanoma showed that GCN2-dependent transcriptional signatures correlated with macrophage polarization, T cell infiltrates, and overall survival. Thus, these data reveal a previously unknown dependence of tumors on myeloid GCN2 signals for protection from immune attack.

PD-1 blockade represents a major therapeutic avenue in anticancer immunotherapy. Delineating mechanisms of secondary resistance to this strategy is increasingly important. Here, we identified the deleterious role of signaling via the type I interferon (IFN) receptor in tumor and antigen presenting cells, that induced the expression of nitric oxide synthase 2 (NOS2), associated with intratumor accumulation of regulatory T cells (Treg) and myeloid cells and acquired resistance to anti-PD-1 monoclonal antibody (mAb). Sustained IFNβ transcription was observed in resistant tumors, in turn inducing PD-L1 and NOS2 expression in both tumor and dendritic cells (DC). Whereas PD-L1 was not involved in secondary resistance to anti-PD-1 mAb, pharmacological or genetic inhibition of NOS2 maintained long-term control of tumors by PD-1 blockade, through reduction of Treg and DC activation. Resistance to immunotherapies, including anti-PD-1 mAb in melanoma patients, was also correlated with the induction of a type I IFN signature. Hence, the role of type I IFN in response to PD-1 blockade should be revisited as sustained type I IFN signaling may contribute to resistance to therapy. CA extern

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.

Pancreatic ductal adenocarcinoma (PDA) has a highly immunosuppressive microenvironment, which is contributed by the complex interaction between cancer cells and a heterogeneous population of stromal cells. Therefore, facile and trackable models are needed for integrative and dynamic interrogation of cancer-stroma interaction. Here, we tracked the immunoevolution of PDA in a genetically-defined transplantable model of mouse pancreatic tumour organoids that recapitulates the progression of the disease from early preinvasive lesions to metastatic carcinomas. We demonstrated that organoid-derived isografts (ODI) can be used as a biological source of biomarkers (NT5E, TGFB1, FN1, and ITGA5) of aggressive molecular subtypes of human PDA. In ODI, infiltration from leukocytes is an early event during progression of the disease as observed for autochthonous models. Neoplastic progression was associated to accumulation of Maf+ macrophages, which inversely correlated with CD8+ T cells infiltration. Consistently, levels of MAF were enriched in human PDA subtypes characterized by abundance of macrophage-related transcripts and indicated poor patients’ survival. Density of MAF+ macrophages was higher in human PDA tissues compared to preinvasive lesions. Our results suggest that ODIs represent a suitable system for genotypic-immunophenotypic studies and support the hypothesis of MAF+ macrophages as a prominent immunosuppressive population in PDA.

Myeloid-Derived Suppressor Cells (MDSCs), immunosuppressive cells that promote tumor growth, represent an attractive target in cancer immunotherapy. However, the clinical success of this strategy is limited by the lack of efficient drug delivery vehicles targeting this cell compartment. The objective of this work was to develop a delivery carrier, multilayer polymer nanocapsules, with the capacity to co-encapsulate two types of immunomodulatory drugs, a chemokine and an RNAi sequence, aimed at reverting MDSC-mediated immunosuppression. The chemokine CCL2, intended to attract monocyte-macrophage MDSCs, was encapsulated within the L2 inverse micellar aqueous domains of the lipid core of these nanocapsules. On the other hand, two different RNAi sequences that modulate the CCAAT/enhancer-binding protein beta (C/EBPβ) pathway, shC/EBPβ and miR 142-3p, were successfully associated to their polymer shell. These RNAi sequences were covered by subsequent layers of polyarginine and hyaluronic acid, thereby creating multi-layered assemblies that protected them and facilitated their targeted delivery. The in vitro studies performed in primary MDSCs cultures showed the capacity of miR 142-3p-loaded nanocapsules to reduce the highly immunosuppressive monocyte- macrophage subset. Additionally, the encapsulation of CCL2 within the nanocapsules induced a potent monocyte-macrophage chemoattraction that could be used to direct the therapy to these cell subsets. Finally, in vitro and in vivo studies showed the capacity of shC/EBPβ-loaded nanocapsules to downregulate C/EBPβ levels in MDSCs and to reduce monocyte differentiation into tumor-associated macrophages in an MCA-203 fibrosarcoma mice model. In conclusion, the multilayer polymer nanocapsules described here are efficient vehicles for the co-delivery of proteins and RNA, and are potential candidates as nanomedicines for the modulation of MDSCs. This work was supported by LYMPHOTARG (ERA-NET EuroNanoMed ProgramISCIII, ref PS09/02670) and NICHE projects (ERA-NET EuroNanoMed II framework by ISCIII through CIBER- BBN), which received funding from the European Union's Seventh Framework Programme. The SAXS measurements were completed on the SAXS/WAXS beamLine at the Australian Synchrotron. A. M. L. was supported by a FPU fellowship from the Spanish Ministry of Education SI

Primary or secondary resistance to immune checkpoint therapy is often analyzed in primary tumors rather than metastases. In this issue of Cell, Jiao et al. show that the site of metastatic diffusion matters and bones empower a particularly hostile environment.

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.

Cancer cells hijack tumor-associated macrophages to sustain their outgrowth. In this issue, Mougiakakos and colleagues identify the IKZF1–IRF4/IRF5 axis as a key molecular pathway regulating macrophage polarization in multiple myeloma. These results suggest targeting IKZF1 could provide a new strategy to reprogram myeloma-associated macrophages toward a tumoricidal and immune-activating phenotype. See article by Mougiakakos et al., p. 265

The expression of Prostate Specific-Membrane Antigen (PSMA) increases in high-grade prostate carcinoma envisaging a role in growth and progression. We show here that clustering PSMA at LNCaP or PC3-PSMA cell membrane activates AKT and MAPK pathways thus promoting proliferation and survival. PSMA activity was dependent on the assembly of a macromolecular complex including filamin A, beta1 integrin, p130CAS, c-Src and EGFR. Within this complex beta1 integrin became activated thereby inducing a c-Src-dependent EGFR phosphorylation at Y1086 and Y1173 EGF-independent residues. Silencing or blocking experiments with drugs demonstrated that all the complex components were required for full PSMA-dependent promotion of cell growth and/or survival in 3D culture, but that p130CAS and EGFR exerted a major role. All PSMA complex components were found assembled in multiple samples of two high-grade prostate carcinomas and associated with EGFR phosphorylation at Y1086. The expression of p130CAS and pEGFRY1086 was thus analysed by tissue micro array in 16 castration-resistant prostate carcinomas selected from 309 carcinomas and stratified from GS 3+4 to GS 5+5. Patients with Gleason Score ≤5 resulted negative whereas those with GS≥5 expressed p130CAS and pEGFRY1086 in 75% and 60% of the cases, respectively. Collectively, our results demonstrate for the first time that PSMA recruits a functionally active complex which is present in high-grade patients. In addition, two components of this complex, p130CAS and the novel pEGFRY1086, correlate with progression in castration-resistant patients and could be therefore useful in therapeutic or surveillance strategies of these patients.

Effective immunotherapy, whether by checkpoint blockade or adoptive cell therapy, is limited in most patients by a key barrier: the immunosuppressive tumor microenvironment. Suppression of tumor-specific T cells is orchestrated by the activity of a variety of stromal myeloid and lymphoid cells. These often display inducible suppressive mechanisms that are triggered by the same anti-tumor inflammatory response that the immunotherapy intends to create. Therefore, a more comprehensive understanding of how the immunosuppressive milieu develops and persists is critical in order to harness the full power of immunotherapy of cancer.

Telomerase (TERT) is overexpressed in 80% to 90% of primary tumors and contributes to sustaining the transformed phenotype. The identification of several TERT epitopes in tumor cells has elevated the status of TERT as a potential universal target for selective and broad adoptive immunotherapy. TERT-specific cytotoxic T lymphocytes (CTL) have been detected in the peripheral blood of B-cell chronic lymphocytic leukemia (B-CLL) patients, but display low functional avidity, which limits their clinical utility in adoptive cell transfer approaches. To overcome this key obstacle hindering effective immunotherapy, we isolated an HLA-A2–restricted T-cell receptor (TCR) with high avidity for human TERT from vaccinated HLA-A*0201 transgenic mice. Using several relevant humanized mouse models, we demonstrate that TCR-transduced T cells were able to control human B-CLL progression in vivo and limited tumor growth in several human, solid transplantable cancers. TERT-based adoptive immunotherapy selectively eliminated tumor cells, failed to trigger a self–MHC-restricted fratricide of T cells, and was associated with toxicity against mature granulocytes, but not toward human hematopoietic progenitors in humanized immune reconstituted mice. These data support the feasibility of TERT-based adoptive immunotherapy in clinical oncology, highlighting, for the first time, the possibility of utilizing a high-avidity TCR specific for human TERT. Cancer Res; 76(9); 2540–51. ©2016 AACR.

This unit presents methods to assess the immunosuppressive properties of immunoregulatory cells of myeloid origin, such as myeloid-derived suppressor cells (MDSCs), both in vitro and in vivo in mice, as well as in biological samples from cancer patients. These methods could be adapted to test the impact of different suppressive populations on T cell activation, proliferation, and cytotoxic activity; moreover, they could be useful to assess the influence exerted by genetic modifications, chemical inhibitors, and drugs on immune suppressive pathways © 2018 by John Wiley & Sons, Inc.

Shwachman-Diamond syndrome (SDS) is one of the more common inherited bone marrow failure syndromes, characterized by neutropenia, occasional thrombocytopenia, and anemia. Bone marrow evaluation reveals an increased number of monocytes and mature B cells along with decreased granulocytes. However, little is known about the subpopulations of peripheral blood cells, and few previous publications have been based on a small number of patients. Here, we report a comprehensive immunophenotypic analysis from a cohort of 37 SDS patients who display impairment mostly in the myeloid compartment with a deficiency also in the number of B cells and CD4/CD8 double-negative T cells.

Critical immune-suppressive pathways beyond programmed death 1 (PD-1) and programmed death ligand 1 (PD-L1) require greater attention. Nectins and nectin-like molecules might be promising targets for immunotherapy, since they play critical roles in cell proliferation and migration and exert immunomodulatory functions in pathophysiological conditions. Here, we show CD155 expression in both malignant cells and tumor-infiltrating myeloid cells in humans and mice. Cd155–/– mice displayed reduced tumor growth and metastasis via DNAM-1 upregulation and enhanced effector function of CD8+ T and NK cells, respectively. CD155-deleted tumor cells also displayed slower tumor growth and reduced metastases, demonstrating the importance of a tumor-intrinsic role of CD155. CD155 absence on host and tumor cells exerted an even greater inhibition of tumor growth and metastasis. Blockade of PD-1 or both PD-1 and CTLA4 was more effective in settings in which CD155 was limiting, suggesting the clinical potential of cotargeting PD-L1 and CD155 function.

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., Signaling and transcriptional regulation of MDSC activity remains largely undefined. Here the authors show that monocytic MDSC immunosuppression is triggered by c-FLIP and requires NFκB, implicate this axis in cancer prognosis and response to therapy, and employ ectopic FLIP to treat immunopathology.

The roles of tumor-associated macrophages (TAMs) and circulating monocytes in human cancer are poorly understood. Here, we show that monocyte subpopulation distribution and transcriptomes are significantly altered by the presence of endometrial and breast cancer. Furthermore, TAMs from endometrial and breast cancers are transcriptionally distinct from monocytes and their respective tissue-resident macrophages. We identified a breast TAM signature that is highly enriched in aggressive breast cancer subtypes and associated with shorter disease-specific survival. We also identified an auto-regulatory loop between TAMs and cancer cells driven by tumor necrosis factor alpha involving SIGLEC1 and CCL8, which is self-reinforcing through the production of CSF1. Together these data provide direct evidence that monocyte and macrophage transcriptional landscapes are perturbed by cancer, reflecting patient outcomes.

Myeloid-derived suppressor cells (MDSCs) are a naturally occurring immune regulatory population associated with inhibition of ongoing inflammatory responses. In vitro generation of MDSCs from bone marrow has been shown to enhance survival in an acute model of lethal graft-versus-host disease (GVHD). However, donor MDSC infusion only partially ameliorates GVHD lethality. In order to improve the potential therapeutic benefit and ultimately survival outcomes, we set out to investigate the fate of MDSCs after transfer in the setting of acute GVHD (aGVHD). MDSCs transferred to lethally irradiated recipients of allogeneic donor hematopoietic grafts are exposed to an intense inflammatory environment associated with aGVHD, which we now show directly undermines their suppressive capacity. Under a conditioning regimen and GVHD inflammatory settings, MDSCs rapidly lose suppressor function and their potential to inhibit GVHD lethality, which is associated with their induced conversion toward a mature inflammasome-activated state. We find even brief in vitro exposure to inflammasome-activating mediators negates the suppressive potential of cultured murine and human-derived MDSCs. Consistent with a role for the inflammasome, donor MDSCs deficient in the adaptor ASC (apoptosis-associated speck-like protein containing a CARD), which assembles inflammasome complexes, conferred improved survival of mice developing GVHD compared with wild-type donor MDSCs. These data suggest the use of MDSCs as a therapeutic approach for preventing GVHD and other systemic inflammatory conditions will be more effective when combined with approaches limiting in vivo MDSC inflammasome activation, empowering MDSCs to maintain their suppressive potential.

Novel models of autochthonous tumorigenesis and adoptive T cell therapy (ATT) are providing new clues regarding the pro-tumorigenic and immunosuppressive effects of myeloid-derived suppressor cells (MDSC), and their interaction with T cells. New findings are shifting the perception of the main level at which MDSC act, from direct cell-to-cell suppression to others, such as limiting T cell infiltration. Adoptively transferred, high-avidity T cells recognizing peptides with high-affinity for MHC-I eliminated large tumors. However, low-avidity T cells or low-affinity peptides resulted in failure to eradicate tumors. Manipulation of intratumoral myeloid cells improved the outcome of otherwise unsuccessful ATT. Therefore, therapeutic intervention directed at the tumor stroma might be required when using suboptimal T cells for ATT.

In this issue of Cancer Cell, Halama et al. (2016) further advance chemokine interference as a therapeutic option for cancer by demonstrating the effect of CCR5 blockade in reshaping macrophage polarization toward an anti-tumor functional state in patient-derived tumor models and liver metastases of colorectal cancer patients.

Bone marrow microenvironment is fundamental for hematopoietic homeostasis. Numerous efforts have been made to reproduce or manipulate its activity to facilitate engraftment after hematopoietic stem cell transplantation but clinical results remain unconvincing. This probably reflects the complexity of the hematopoietic niche. Recent data have demonstrated the fundamental role of stromal and myeloid cells in regulating hematopoietic stem cell self-renewal and mobilization in the bone marrow. In this study we unveil a novel interaction by which bone marrow mesenchymal stromal cells induce the rapid differentiation of CD11b+ myeloid cells from bone marrow progenitors. Such an activity requires the expression of nitric oxide synthase-2. Importantly, the administration of these mesenchymal stromal cell-educated CD11b+ cells accelerates hematopoietic reconstitution in bone marrow transplant recipients. We conclude that the liaison between mesenchymal stromal cells and myeloid cells is fundamental in hematopoietic homeostasis and suggests that it can be harnessed in clinical transplantation.

Oncogenes can aid tumor progression in a cancer cell-extrinsic way. In this issue of Immunity, Glodde et al. (2017) demonstrate that interference with c-MET tyrosine kinase receptor can relieve neutrophil-dependent immune suppression and unleash the effectiveness of immunotherapy even in the context of c-MET-independent tumors.