Learning from TCR Signaling and Immunological Synapse Assembly to Build New Chimeric Antigen Receptors (CARs).

Chimeric antigen receptor (CAR) T cell immunotherapy is a revolutionary pillar in cancer treatment. Clinical experience has shown remarkable successes in the treatment of certain hematological malignancies but only limited efficacy against B cell chronic lymphocytic leukemia (CLL) and other cancer types, especially solid tumors. A wide range of engineering strategies have been employed to overcome the limitations of CAR T cell therapy. However, it has become increasingly clear that CARs have unique, unexpected features; hence, a deep understanding of how CARs signal and trigger the formation of a non-conventional immunological synapse (IS), the signaling platform required for T cell activation and execution of effector functions, would lead a shift from empirical testing to the rational design of new CAR constructs. Here, we review current knowledge of CARs, focusing on their structure, signaling and role in CAR T cell IS assembly. We, moreover, discuss the molecular features accounting for poor responses in CLL patients treated with anti-CD19 CAR T cells and propose CLL as a paradigm for diseases connected to IS dysfunctions that could significantly benefit from the development of novel CARs to generate a productive anti-tumor response

Glycerophosphoinositol 4-phosphate, a putative endogenous inhibitor of adenylylcyclase.

In a continuous line of rat thyroid cells transformed by the k-ras oncogene (KiKi), the expression of ras-p21 correlates with an increased activity of a phosphoinositide-specific phospholipase A2, which leads to elevated levels of glycerophosphoinositols. In this study we have characterized the biological activities of these compounds. Growth and differentiation in thyroid cells are mainly regulated by the activation of adenylylcyclase. Therefore, we have studied the effects of glycerophosphoinositols on the activity of this enzyme using a normal thyroid cell line (FRTL5). Micromolar concentrations of glycerophosphoinositol 4-phosphate (GroPIns-4-P) caused a approximately 50% inhibition of the adenylylcyclase activity in FRTL5 membranes stimulated by the GTP-binding protein activator fluoroaluminate. Similar concentrations of GroPIns-4-P were detected in KiKi cells but not in the normal FRTL5 line. Micromolar GroPIns-4-P was found to be taken up by intact FRTL5 cells and to induce nearly 50% inhibition of the thyrotropin- and cholera toxin-induced increase in cAMP levels. Similar results were also observed in other cell lines (smooth muscle, pituitary cells, and pneumocytes). GroPIns-4-P inhibited cAMP-dependent cellular functions such as iodide uptake and thymidine incorporation in FRTL5 cells when stimulated by thyrotropin and cholera toxin but not when induced by forskolin. These results are consistent with GroPIns-4-P exerting an inhibitory effect on the GTP-binding protein that stimulates adenylycyclase. We propose that GroPIns-4-P might mediate a mechanism of cross-talk between adenylylcyclase and phospholipase A2 in thyroid as well as in other cell systems

Effects of Intracellular Calcium and Actin Cytoskeleton on TCR Mobility Measured by Fluorescence Recovery

Background: The activation of T lymphocytes by specific antigen is accompanied by the formation of a specialized signaling region termed the immunological synapse, characterized by the clustering and segregation of surface molecules and, in particular, by T cell receptor (TCR) clustering. Methodology/Principal Findings: To better understand TCR motion during cellular activation, we used confocal microscopy and photo-bleaching recovery techniques to investigate the lateral mobility of TCR on the surface of human T lymphocytes under various pharmacological treatments. Using drugs that cause an increase in intracellular calcium, we observed a decrease in TCR mobility that was dependent on a functional actin cytoskeleton. In parallel experiments measurement of filamentous actin by FACS analysis showed that raising intracellular calcium also causes increased polymerization of the actin cytoskeleton. These in vitro results were analyzed using a mathematical model that revealed effective binding parameters between TCR and the actin cytoskeleton. Conclusion/Significance: We propose, based on our results, that increase in intracellular calcium levels leads to actin polymerization and increases TCR/cytoskeleton interactions that reduce the overall mobility of the TCR. In a physiological setting, this may contribute to TCR re-positioning at the immunological synapse

Kartezio: Evolutionary Design of Explainable Pipelines for Biomedical Image Analysis

An unresolved issue in contemporary biomedicine is the overwhelming number and diversity of complex images that require annotation, analysis and interpretation. Recent advances in Deep Learning have revolutionized the field of computer vision, creating algorithms that compete with human experts in image segmentation tasks. Crucially however, these frameworks require large human-annotated datasets for training and the resulting models are difficult to interpret. In this study, we introduce Kartezio, a modular Cartesian Genetic Programming based computational strategy that generates transparent and easily interpretable image processing pipelines by iteratively assembling and parameterizing computer vision functions. The pipelines thus generated exhibit comparable precision to state-of-the-art Deep Learning approaches on instance segmentation tasks, while requiring drastically smaller training datasets, a feature which confers tremendous flexibility, speed, and functionality to this approach. We also deployed Kartezio to solve semantic and instance segmentation problems in four real-world Use Cases, and showcase its utility in imaging contexts ranging from high-resolution microscopy to clinical pathology. By successfully implementing Kartezio on a portfolio of images ranging from subcellular structures to tumoral tissue, we demonstrated the flexibility, robustness and practical utility of this fully explicable evolutionary designer for semantic and instance segmentation.Comment: 36 pages, 6 main Figures. The Extended Data Movie is available at the following link: https://www.youtube.com/watch?v=r74gdzb6hdA. The source code is available on Github: https://github.com/KevinCortacero/Kartezi

Il falso dilemma pubblico-privato. L’anomalia della scuola italiana nel contesto europeo

Il modello organizzativo del sistema scolastico italiano in prospettiva storica e comparata nel contesto europeo; prospettive per un adeguamento della scuola italiana al “sistema medio europeo”.- Indice #4- Introduzione, Marcello Pacini #8- Nota metodologica #14- Cap.I Il modello organizzativo del sistema scolastico italiano #18- Cap.II Il sistema medio europeo. Sistemi e problemi di un'analisi comparata #54- Cap.III Verso la deburocratizzazione. Prospettive per un adeguamento della scuola italiana al “sistema medio europeo” #114- Allegato I La struttura del sistema scolastico italiano #146- Relazioni e interventi di discussione sulla ricerca #206- Intervento On. Francesco Casati, Presidente della Commissione Istruzione e Belle Arti della Camera dei Deputati #208- Intervento Sen. Luigi Covatta, Sottosegretario di Stato alla Pubblica Istruzione #212- Intervento Sen. Salvatore Valitutti, Presidente della Commissione Istruzione Pubblica e Belle Arti del Senato della Repubblica #215- Intervento Emanuele Caruso, Dirigente Generale dell'Istruzione Tecnica, Ministero della Pubblica Istruzione #219- Intervento Mario Dupuis, Responsabile Scuola del Movimento Popolare #222- Intervento On. Laura Fincato, Vicepresidente della Commissione Istruzione e Belle Arti della Camera dei Deputati #225- Intervento Aureliana Alberici, Responsabile Scuola/Università della Direzione del P.C.I. #229- Intervento Paolo Serreri, Federazione Scuola Università C.G.I.L. #237- Intervento Daniela Silvestri, Rappresentante nazionale del Sindacato Nazionale Autonomo Lavoratori Scuola SNALS #242- Intervento Paolo Martelli, Direttore di POLITEIA - Centro per la ricerca e la formazione in politica ed etica #245- Intervento Salvatore Sechi, Professore ordinario di Storia Contemporanea, Università di Bologna #252- Intervento Piero Romei, Ricercatore dell'ISGO #257- Intervento Giovanni Bechelloni, Professore ordinario di Sociologia dei processi culturali, Università di Firenze #263- Intervento Giorgio Allulli, Censis #266- Intervento Graziella Morselli, FNISM #270- Intervento Luigi Pedrazzi, Il Mulino #274- Intervento Luciano Benadusi, Responsabile Settore Università e ricerca scientifica della Direzione Socialista #277- Intervento Mario Caronna #Coordinatore scientifico del Centro Studi di Milano #282- Intervento Adriana Rosas, Ricercatore presso il CLAS #287- Intervento Giovanni Tesoro, Ricercatore presso l’ISGO #290- Considerazioni conclusive Luisa Ribolzi, CLAS #29

Stepwise Maturation of Lytic Granules during Differentiation and Activation of Human CD8+ T Lymphocytes

During differentiation, cytotoxic T lymphocytes (CTL) acquire their killing potential through the biogenesis and maturation of lytic granules that are secreted upon target cell recognition. How lytic granule load in lytic molecules evolves during CTL differentiation and which subsets of lytic granules are secreted following activation remains to be investigated. We set up a flow cytometry approach to analyze single lytic granules isolated from primary human CTL according to their size and molecular content. During CTL in vitro differentiation, a relatively homogeneous population of lytic granules appeared through the progressive loading of Granzyme B, Perforin and Granzyme A within LAMP1+ lysosomes. PMA/ionomycin-induced lytic granule exocytosis was preceded by a rapid association of the docking molecule Rab27a to approximately half of the lytic granules. Activated CTL were found to limit exocytosis by sparing lytic granules including some associated to Rab27a. Our study provides a quantification of key steps of lytic granule biogenesis and highlights the potential of flow cytometry to study organelle composition and dynamics

Etude de la dynamique moléculaire au site de contact entre les lymphocytes T et les cellules présentatrices de l'antigène

TOULOUSE3-BU Sciences (315552104) / SudocSudocFranceF

Influence des lymphocytes T CD4+ CD25+ régulateurs sur la dynamique de formation de la synapse immunologique entre un lymphocyte T CD4+ effecteur et une cellule présentatrice d'antigène

TOULOUSE3-BU Sciences (315552104) / SudocSudocFranceF