Dissecting Treg Heterogeneity in Human Cancer to find Novel Targets of Immunotheraphy

T cell dysfunction in the tumor microenvironment (TME) mediated by the hyper-activation of inhibitory subpopulations such as regulatory T cells (Treg) represents a major obstacle to effective antitumor immunity and immunotherapy. The molecular mechanisms at the basis of the increased immunosuppressive capacity of Treg cells in the TME are currently not clear. Preclinical data shows that bulk Treg depletion improves T cell infiltration and its antitumor activity but simultaneously results in deleterious autoimmune responses. Targeting hyperactive Treg specifically present in the tumor would improve antitumor immunity while reducing toxicity. To this aim, we developed 30-parameter FACS to profile millions of single T cells from chemotherapy-naïve cancer patients. Computational algorithms applied to single-cell data identified T cell subsets preferentially enriched in tumors compared to adjacent normal lung tissue and peripheral blood. We found that the Interferon regulatory factor 4 (IRF4) defines a novel specific subpopulation of tumor infiltrating Tregs with superior immunosuppressive capacity. Integration of transcriptomic data and whole genome binding sites of transcription factors further revealed that IRF4, either alone or in combination with its partner BATF, directly controlled a molecular program responsible for Treg hyperproliferation and suppressive functions that included CTLA-4, ICOS, TNFRSF9 (4-1BB) and IL1R2. Accordingly, deletion of Irf4 exclusively in Tregs resulted in delayed tumor growth in mice while the abundance of IRF4+ Tregs correlated with poor prognosis in patients with multiple human cancers. We further identify mesenchyme homeobox 1 (MEOX1), a transcription factor novel to Treg biology, as sufficient to reprogram circulating Tregs to a tumor-infiltrating 4-1BB+ phenotype. MEOX1 activity sustains hyper-activated Treg gene expression and immunosuppression by favoring chromatin accessibility at sites regulated by AP-1/IRF4/BATF. Thus, Interfering with IRF4-dependent molecular program, which is shared by several solid tumors, may represent a novel immunotherapeutic approach capable to block immunosuppression in the tumor microenvironment

Regulation of the RNA-binding protein Smaug by the GPCR Smoothened via the kinase Fused

From fly to mammals, the Smaug/Samd4 family of prion-like RNA-binding proteins control gene expression by destabilizing and/or repressing the translation of numerous target transcripts. However, the regulation of its activity remains poorly understood. We show that Smaug's protein levels and mRNA repressive activity are downregulated by Hedgehog signaling in tissue culture cells. These effects rely on the interaction of Smaug with the G-protein coupled receptor Smoothened, which promotes the phosphorylation of Smaug by recruiting the kinase Fused. The activation of Fused and its binding to Smaug are sufficient to suppress its ability to form cytosolic bodies and to antagonize its negative effects on endogenous targets. Importantly, we demonstrate in vivo that HH reduces the levels of smaug mRNA and increases the level of several mRNAs downregulated by Smaug. Finally, we show that Smaug acts as a positive regulator of Hedgehog signaling during wing morphogenesis. These data constitute the first evidence for a post-translational regulation of Smaug and reveal that the fate of several mRNAs bound to Smaug is modulated by a major signaling pathway.Fil: Bruzzone, Lucia. Centre National de la Recherche Scientifique; Francia. Universite de Paris; FranciaFil: Argüelles, Camilla. Centre National de la Recherche Scientifique; Francia. Universite de Paris; FranciaFil: Sanial, Matthieu. Universite de Paris; Francia. Centre National de la Recherche Scientifique; FranciaFil: Miled, Samia. Universite de Paris; Francia. Centre National de la Recherche Scientifique; FranciaFil: Alvisi, Giorgia. Universite de Paris; Francia. Centre National de la Recherche Scientifique; FranciaFil: Gonçalves Antunes, Marina. Universite de Paris; Francia. Centre National de la Recherche Scientifique; FranciaFil: Qasrawi, Fairouz. Universite de Paris; Francia. Centre National de la Recherche Scientifique; FranciaFil: Holmgren, Robert A.. Northwestern University; Estados UnidosFil: Smibert, Craig A. University of Toronto; CanadáFil: Lipshitz, Howard D.. University of Toronto; CanadáFil: Boccaccio, Graciela Lidia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Plessis, Anne. Centre National de la Recherche Scientifique; Francia. Universite de Paris; FranciaFil: Bécam, Isabelle. Centre National de la Recherche Scientifique; Francia. Universite de Paris; Franci

Acquisition of suppressive function by conventional T cells limits antitumor immunity upon Treg depletion

Regulatory T (Treg) cells contribute to immune homeostasis but suppress immune responses to cancer. Strategies to disrupt Treg cell–mediated cancer immunosuppression have been met with limited clinical success, but the underlying mechanisms for treatment failure are poorly understood. By modeling Treg cell–targeted immunotherapy in mice, we find that CD4+ Foxp3− conventional T (Tconv) cells acquire suppressive function upon depletion of Foxp3+ Treg cells, limiting therapeutic efficacy. Foxp3− Tconv cells within tumors adopt a Treg cell–like transcriptional profile upon ablation of Treg cells and acquire the ability to suppress T cell activation and proliferation ex vivo. Suppressive activity is enriched among CD4+ Tconv cells marked by expression of C-C motif receptor 8 (CCR8), which are found in mouse and human tumors. Upon Treg cell depletion, CCR8+ Tconv cells undergo systemic and intratumoral activation and expansion, and mediate IL-10–dependent suppression of antitumor immunity. Consequently, conditional deletion of Il10 within T cells augments antitumor immunity upon Treg cell depletion in mice, and antibody blockade of IL-10 signaling synergizes with Treg cell depletion to overcome treatment resistance. These findings reveal a secondary layer of immunosuppression by Tconv cells released upon therapeutic Treg cell depletion and suggest that broader consideration of suppressive function within the T cell lineage is required for development of effective Treg cell–targeted therapies

Two subsets of stem-like CD8+ memory T cell progenitors with distinct fate commitments in humans

T cell memory relies on the generation of antigen-specific progenitors with stem-like properties. However, the identity of these progenitors has remained unclear, precluding a full understanding of the differentiation trajectories that underpin the heterogeneity of antigen-experienced T cells. We used a systematic approach guided by single-cell RNA-sequencing data to map the organizational structure of the human CD8+ memory T cell pool under physiological conditions. We identified two previously unrecognized subsets of clonally, epigenetically, functionally, phenotypically and transcriptionally distinct stem-like CD8+ memory T cells. Progenitors lacking the inhibitory receptors programmed death-1 (PD-1) and T cell immunoreceptor with Ig and ITIM domains (TIGIT) were committed to a functional lineage, whereas progenitors expressing PD-1 and TIGIT were committed to a dysfunctional, exhausted-like lineage. Collectively, these data reveal the existence of parallel differentiation programs in the human CD8+ memory T cell pool, with potentially broad implications for the development of immunotherapies and vaccines

What is the role of the placebo effect for pain relief in neurorehabilitation? Clinical implications from the Italian consensus conference on pain in neurorehabilitation

Background: It is increasingly acknowledged that the outcomes of medical treatments are influenced by the context of the clinical encounter through the mechanisms of the placebo effect. The phenomenon of placebo analgesia might be exploited to maximize the efficacy of neurorehabilitation treatments. Since its intensity varies across neurological disorders, the Italian Consensus Conference on Pain in Neurorehabilitation (ICCP) summarized the studies on this field to provide guidance on its use. Methods: A review of the existing reviews and meta-analyses was performed to assess the magnitude of the placebo effect in disorders that may undergo neurorehabilitation treatment. The search was performed on Pubmed using placebo, pain, and the names of neurological disorders as keywords. Methodological quality was assessed using a pre-existing checklist. Data about the magnitude of the placebo effect were extracted from the included reviews and were commented in a narrative form. Results: 11 articles were included in this review. Placebo treatments showed weak effects in central neuropathic pain (pain reduction from 0.44 to 0.66 on a 0-10 scale) and moderate effects in postherpetic neuralgia (1.16), in diabetic peripheral neuropathy (1.45), and in pain associated to HIV (1.82). Moderate effects were also found on pain due to fibromyalgia and migraine; only weak short-term effects were found in complex regional pain syndrome. Confounding variables might have influenced these results. Clinical implications: These estimates should be interpreted with caution, but underscore that the placebo effect can be exploited in neurorehabilitation programs. It is not necessary to conceal its use from the patient. Knowledge of placebo mechanisms can be used to shape the doctor-patient relationship, to reduce the use of analgesic drugs and to train the patient to become an active agent of the therapy

What is the role of the placebo effect for pain relief in neurorehabilitation? Clinical implications from the Italian Consensus Conference on Pain in Neurorehabilitation

Background: It is increasingly acknowledged that the outcomes of medical treatments are influenced by the context of the clinical encounter through the mechanisms of the placebo effect. The phenomenon of placebo analgesia might be exploited to maximize the efficacy of neurorehabilitation treatments. Since its intensity varies across neurological disorders, the Italian Consensus Conference on Pain in Neurorehabilitation (ICCP) summarized the studies on this field to provide guidance on its use. Methods: A review of the existing reviews and meta-analyses was performed to assess the magnitude of the placebo effect in disorders that may undergo neurorehabilitation treatment. The search was performed on Pubmed using placebo, pain, and the names of neurological disorders as keywords. Methodological quality was assessed using a pre-existing checklist. Data about the magnitude of the placebo effect were extracted from the included reviews and were commented in a narrative form. Results: 11 articles were included in this review. Placebo treatments showed weak effects in central neuropathic pain (pain reduction from 0.44 to 0.66 on a 0-10 scale) and moderate effects in postherpetic neuralgia (1.16), in diabetic peripheral neuropathy (1.45), and in pain associated to HIV (1.82). Moderate effects were also found on pain due to fibromyalgia and migraine; only weak short-term effects were found in complex regional pain syndrome. Confounding variables might have influenced these results. Clinical implications: These estimates should be interpreted with caution, but underscore that the placebo effect can be exploited in neurorehabilitation programs. It is not necessary to conceal its use from the patient. Knowledge of placebo mechanisms can be used to shape the doctor-patient relationship, to reduce the use of analgesic drugs and to train the patient to become an active agent of the therapy

Development, application and computational analysis of high-dimensional fluorescent antibody panels for single-cell flow cytometry

The interrogation of single cells is revolutionizing biology, especially our understanding of the immune system. Flow cytometry is still one of the most versatile and high-throughput approaches for single-cell analysis, and its capability has been recently extended to detect up to 28 colors, thus approaching the utility of cytometry by time of flight (CyTOF). However, flow cytometry suffers from autofluorescence and spreading error (SE) generated by errors in the measurement of photons mainly at red and far-red wavelengths, which limit barcoding and the detection of dim markers. Consequently, development of 28-color fluorescent antibody panels for flow cytometry is laborious and time consuming. Here, we describe the steps that are required to successfully achieve 28-color measurement capability. To do this, we provide a reference map of the fluorescence spreading errors in the 28-color space to simplify panel design and predict the success of fluorescent antibody combinations. Finally, we provide detailed instructions for the computational analysis of such complex data by existing, popular algorithms (PhenoGraph and FlowSOM). We exemplify our approach by designing a high-dimensional panel to characterize the immune system, but we anticipate that our approach can be used to design any high-dimensional flow cytometry panel of choice. The full protocol takes a few days to complete, depending on the time spent on panel design and data analysis

CRUSTY: a versatile web platform for the rapid analysis and visualization of high-dimensional flow cytometry data

Abstract Flow cytometry (FCM) can investigate dozens of parameters from millions of cells and hundreds of specimens in a short time and at a reasonable cost, but the amount of data that is generated is considerable. Computational approaches are useful to identify novel subpopulations and molecular biomarkers, but generally require deep expertize in bioinformatics and the use of different platforms. To overcome these limitations, we introduce CRUSTY, an interactive, user-friendly webtool incorporating the most popular algorithms for FCM data analysis, and capable of visualizing graphical and tabular results and automatically generating publication-quality figures within minutes. CRUSTY also hosts an interactive interface for the exploration of results in real time. Thus, CRUSTY enables a large number of users to mine complex datasets and reduce the time required for data exploration and interpretation. CRUSTY is accessible at https://crusty.humanitas.it/

New Policies and Practises for European Sharing Cities

Il volume raccoglie i risultati del progetto \u201cNew Policies and Practices for European Sharing Cities \u2013EUcity\u201d, attivato nell\u2019ambito dello Jean Monnet Project e co-finanziato dall\u2019Erasmus+ Project dell\u2019Unione Europea. Muovendo dall\u2019assunto per il quale le citt\ue0 sono i principali recettori dei cambiamenti del mercato poich\ue9 si distinguono come legami interconnessi tra la societ\ue0, lo Stato e l\u2019Unione europea, gli Autori hanno analizzato il fenomeno della sharing economy, alcuni modelli di governance e di fornitura di beni e servizi sul mercato. Come illustrato dal nucleo argomentativo dell\u2019opera, infatti, le sfide normative della sharing economy non derivano solo dalla novit\ue0 del modello economico p2p, ma anche da una distribuzione completamente nuova delle responsabilit\ue0 normative tra gli attori coinvolti. A differenza della regolamentazione del mercato tradizionale, la regolamentazione dell\u2019economia della condivisione \ue8 principalmente una questione comunale. Allo stesso tempo, anche il diritto europeo offre spunti di riflessione notevoli. Attualmente, sia gli amministratori locali che gli operatori privati di alcuni settori del mercato (ad es. la casa, i trasporti, etc.), ma anche i cittadini che vogliono sperimentare nuovi servizi, sono obbligati ad agire all\u2019interno di un sistema normativo difficilmente intellegibile. In virt\uf9 delle considerazioni svolte, quindi, il testo apre un fronte critico argomentativo diviso in tre parti: 1) \u201cSharing cities\u201d, in cui si introducono i temi principali relativi ai fenomeni urbani in un contesto di sharing economy; 2) \u201cTrasporti, Turismo e politiche abitative nelle Sharing cities\u201d, contenente un focus riguardante gli aspetti dinamici del vivere in contesti smart; 3) \u201cEsperienze a confronto\u201d, parte dedicata all\u2019analisi empirica delle realt\ue0 sperimentate da Bologna e Barcellona

Single-Cell Sequencing of Mouse Heart Immune Infiltrate in Pressure Overload-Driven Heart Failure Reveals Extent of Immune Activation

BACKGROUND: Inflammation is a key component of cardiac disease, with macrophages and T lymphocytes mediating essential roles in the progression to heart failure. Nonetheless, little insight exists on other immune subsets involved in the cardiotoxic response.METHODS: Here, we used single-cell RNA sequencing to map the cardiac immune composition in the standard murine nonischemic, pressure-overload heart failure model. By focusing our analysis on CD45(+) cells, we obtained a higher resolution identification of the immune cell subsets in the heart, at early and late stages of disease and in controls. We then integrated our findings using multiparameter flow cytometry, immunohistochemistry, and tissue clarification immunofluorescence in mouse and human.RESULTS: We found that most major immune cell subpopulations, including macrophages, B cells, T cells and regulatory T cells, dendritic cells, Natural Killer cells, neutrophils, and mast cells are present in both healthy and diseased hearts. Most cell subsets are found within the myocardium, whereas mast cells are found also in the epicardium. Upon induction of pressure overload, immune activation occurs across the entire range of immune cell types. Activation led to upregulation of key subset-specific molecules, such as oncostatin M in proinflammatory macrophages and PD-1 in regulatory T cells, that may help explain clinical findings such as the refractivity of patients with heart failure to anti-tumor necrosis factor therapy and cardiac toxicity during anti-PD-1 cancer immunotherapy, respectively.CONCLUSIONS: Despite the absence of infectious agents or an autoimmune trigger, induction of disease leads to immune activation that involves far more cell types than previously thought, including neutrophils, B cells, Natural Killer cells, and mast cells. This opens up the field of cardioimmunology to further investigation by using toolkits that have already been developed to study the aforementioned immune subsets. The subset-specific molecules that mediate their activation may thus become useful targets for the diagnostics or therapy of heart failure