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The thymus is a primary lymphoid organ, essential for T cell maturation and selection. There has been long-standing interest in processes underpinning thymus generation and the potential to manipulate it clinically, because alterations of thymus development or function can result in severe immunodeficiency and autoimmunity. Here, we identify epithelial-mesenchymal hybrid cells, capable of long-term expansion in vitro, and able to reconstitute an anatomic phenocopy of the native thymus, when combined with thymic interstitial cells and a natural decellularised extracellular matrix (ECM) obtained by whole thymus perfusion. This anatomical human thymus reconstruction is functional, as judged by its capacity to support mature T cell development in vivo after transplantation into humanised immunodeficient mice. These findings establish a basis for dissecting the cellular and molecular crosstalk between stroma, ECM and thymocytes, and offer practical prospects for treating congenital and acquired immunological diseases. The thymus is essential for T cell maturation and selection, and thymic defects result in severe immune problems. Here the authors identify a thymus cell population that is expandable in vitro, and can repopulate natural thymic matrix to generate tissue that supports mature T cell development in vitro and in vivo.

Defective functionality of thymic epithelial cells (TECs), due to genetic mutations or injuring causes, results in altered T‐cell development, leading to immunodeficiency or autoimmunity. These defects cannot be corrected by hematopoietic stem cell transplantation (HSCT), and thymus transplantation has not yet been demonstrated to be fully curative. Here, we provide proof of principle of a novel approach toward thymic regeneration, involving the generation of thymic organoids obtained by seeding gene‐modified postnatal murine TECs into three‐dimensional (3D) collagen type I scaffolds mimicking the thymic ultrastructure. To this end, freshly isolated TECs were transduced with a lentiviral vector system, allowing for doxycycline‐induced Oct4 expression. Transient Oct4 expression promoted TECs expansion without drastically changing the cell lineage identity of adult TECs, which retain the expression of important molecules for thymus functionality such as Foxn1, Dll4, Dll1, and AIRE. Oct4‐expressing TECs (iOCT4 TEC) were able to grow into 3D collagen type I scaffolds both in vitro and in vivo, demonstrating that the collagen structure reproduced a 3D environment similar to the thymic extracellular matrix, perfectly recognized by TECs. In vivo results showed that thymic organoids transplanted subcutaneously in athymic nude mice were vascularized but failed to support thymopoiesis because of their limited in vivo persistence. These findings provide evidence that gene modification, in combination with the usage of 3D biomimetic scaffolds, may represent a novel approach allowing the use of postnatal TECs for thymic regeneration. Stem Cells Translational Medicine 2019;8:1107–1122 Transient Oct4 expression promoted thymic epithelial cells expansion without drastically changing the cell lineage identity of adult thymic epithelial cells. iOCT4 thymic epithelial cells were able to grow into three‐dimensional collagen type I scaffolds both in vitro and in vivo demonstrating that the collagen structure reproduced a three‐dimensional environment similar to the thymic extracellular matrix, perfectly recognized by thymic epithelial cells. in vivo results showed that thymic organoids transplanted subcutaneously in athymis nude mice were vascularized but failed to support thymopoiesis because of the limited in vivo persistence. These findings provide evidence that gene modification, in combination with the usage of three‐dimensional biomimetic scaffolds, represents a novel approach allowing the use of postnatal thymic epithelial cells for thymic regeneration.

BackgroundAdoptive T cell therapy (ACT) has become a promising option for cancer patients. While tumor-infiltrating lymphocytes were initially exploited as a source of tumor reactive lymphocytes, T cells genetically redirected to the tumor by TCR/CAR gene transfer are now in clinical validation. In the case of solid tumors, unfavorable immunosuppressive microenvironments remain recognized barriers to therapeutic efficacy. We have recently reported that the therapeutic activity of ACT against poorly immunogenic and indolent prostate cancer is improved by the concurrent targeting of the tumor stroma by mean of T cells redirected to an ubiquitously expressed minor histocompatibility antigen or a tumor vessel targeted TNF derivative. We have now taken the concept further and hypothesized that local radiotherapy (RT), might also synergize with ACT by promoting lymphocyte endothelial transmigration and tumor recognition, and ultimately favor abscopal effects.MethodsWe investigated the combination of local RT and ACT in TRAMP (Transgenic Adenocarcinoma of the Mouse Prostate) mice and in mice bearing subcutaneous B16/B16-OVA (MO4) or TRAMP-C2/TRAMP-C2-OVA tumors. Local RT was delivered by X-RAD SmART (the Small Animal Radiation Therapy) microirradiator in single dose or hypo-fractioned regimens. ACT consisted of T cells engineered with tumor-specific TCRs. Immunogenic consequences were analyzed by Real-Time PCR, and flow cytometry (FACS) analyses. Prostate tumor debulking was evaluated by histological analyses.ResultsWe found that local hypofractionated RT and ACT, while individually inefficacious in controlling tumor growth, concurred to the debulking of advanced prostate adenocarcinoma when used in combination in treating TRAMP mice. Mechanistically, exposing isolated tumor cells, or the TRAMP mouse prostate to hypo-fractionated RT regimens induced stronger type-I interferon (IFN-I) responses, when compared to single high dose. Acutely, hypofractionated RT promoted better immune tumor infiltration, among which TCR redirected effector cells.ConclusionsData support feasibility and efficacy of combining hypo-fractionated local RT with ACT in the form of TCR engineered T cells to promote prostate cancer recognition and eradication. Tumor debulking was observed in the absence of treatment-related toxicity. Systemic recirculation of TCR redirected T cells was observed. We are now investigating therapeutic effects at distal (metastatic) sites.AcknowledgementsThe authors acknowledge the support of the Italian Association for Cancer Research (AIRC)Ethics ApprovalThe studies involving animals were approved by The Institutional Ethical Committee (IACUC#999).

Wiskott-Aldrich Syndrome (WAS) is a severe X-linked Primary Immunodeficiency that affects 1-10 out of 1 million male individuals. WAS is caused by mutations in the WAS Protein (WASP) expressing gene that leads to the absent or reduced expression of the protein. WASP is a cytoplasmic protein that regulates the formation of actin filaments in hematopoietic cells. WASP deficiency causes many immune cell defects both in humans and in the WAS murine model, the Was(-/-) mouse. Both cellular and humoral immune defects in WAS patients contribute to the onset of severe clinical manifestations, in particular microthrombocytopenia, eczema, recurrent infections, and a high susceptibility to develop autoimmunity and malignancies. Autoimmune diseases affect from 22 to 72% of WAS patients and the most common manifestation is autoimmune hemolytic anemia, followed by vasculitis, arthritis, neutropenia, inflammatory bowel disease, and IgA nephropathy. Many groups have widely explored immune cell functionality in WAS partially explaining how cellular defects may lead to pathology. However, the mechanisms underlying the occurrence of autoimmune manifestations have not been clearly described yet. In the present review, we report the most recent progresses in the study of immune cell function in WAS that have started to unveil the mechanisms contributing to autoimmune complications in WAS patients.

Wiskott–Aldrich syndrome (WAS) is a rare X-linked primary immunodeficiency caused by the defective expression of the WAS protein (WASP) in hematopoietic cells. It has been shown that dendritic cells (DCs) are functionally impaired in WAS patients and was −/− mice. We have previously demonstrated the efficacy and safety of a murine model of WAS gene therapy (GT), using stem cells transduced with a lentiviral vector (LV). The aim of this study was to investigate whether GT can correct DC defects in was −/− mice. As DCs expressing WASP were detected in the secondary lymphoid organs of the treated mice, we tested the in vitro and in vivo function of bone marrow-derived DCs (BMDCs). The BMDCs showed efficient in vitro uptake of latex beads and Salmonella typhimurium . When BMDCs from the treated mice (GT BMDCs) and the was −/− mice were injected into wild-type hosts, we found a higher number of cells that had migrated to the draining lymph nodes compared with mice injected with was −/− BMDCs. Finally, we found that ovalbumin (OVA)-pulsed GT BMDCs or vaccination of GT mice with anti-DEC205 OVA fusion protein can efficiently induce antigen-specific T-cell activation in vivo . These findings show that WAS GT significantly improves DC function, thus adding new evidence of the preclinical efficacy of LV-mediated WAS GT.

The human receptor for advanced glycation endproducts (RAGE) is a multiligand cell surface protein belonging to the immunoglobulin superfamily, and is involved in inflammatory and immune responses. Most importantly, RAGE is considered a receptor for HMGB1 and several S100 proteins, which are Damage-Associated Molecular Pattern molecules (DAMPs) released during tissue damage. In this study we show that the Ager gene coding for RAGE first appeared in mammals, and is closely related to other genes coding for cell adhesion molecules (CAMs) such as ALCAM, BCAM and MCAM that appeared earlier during metazoan evolution. RAGE is expressed at very low levels in most cells, but when expressed at high levels, it mediates cell adhesion to extracellular matrix components and to other cells through homophilic interactions. Our results suggest that RAGE evolved from a family of CAMs, and might still act as an adhesion molecule, in particular in the lung where it is highly expressed or under pathological conditions characterized by an increase of its protein levels.

Species distribution models are powerful tools to infer ecology and support management of conservation and socio‐economic valuable taxa, such as brown trout (Salmo trutta complex). Using a random forest approach, we modelled its distribution in central Italy watercourses, using recent presences/absences and eight environmental/bioclimatic predictors. The model shows (i) high predictive ability (K = 0.76), (ii) predicts suitable, naturally‐infrequent lowland watercourses where brown trout occurs or may occur. Moreover, the prediction values (iii) expresses a remarkable positive monotone relationship with abundance classes of brown trout computed during field sampling, despite such information was not included in the model development. Predictors' importance pointed out to the crucial role of bioclimatic constraints (linked to thermal suitability and habitat availability) over anthropogenic disturbance and lithotypes. This modelling exercise reiterates the importance of modelling approaches based on spatially explicit proxies of species habitat requirements to assist taxa management by revealing suitable but infrequent and singular areas that could be considered worthy of protection. Using freely available spatially explicit environmental‐bioclimatic predictors, in a Random Forest framework, we: (i) accurately modelled the spatial distribution of Salmo trutta complex in central Italy; (ii) revealed the suitability of naturally infrequent lowland watercourses where trout occurrence is only partly known; (iii) provided information on relative contributions of predictors; (iv) detected a relationship between model predictions and brown trout abundance classes.

Introduction School-based comprehensive sexuality education (CSE) is a powerful tool that provides young people with information on all aspects of sexuality and is aimed at protecting their sexual and reproductive health and well-being throughout their lives. Currently, CSE is not integrated within the schools’ curriculum in Italy. This study describes the co-construction, implementation, and evaluation of a CSE project piloted among students attending lower secondary schools, in four regions of Italy. Evidence-based evaluation will be helpful in promote the inclusion of CSE programs in the Italian schools’ curriculum. Methods The pilot scheme was co-constructed by a multidisciplinary curriculum development group through a Delphi process, including educators who conducted the activities. The evaluation followed three directions: the program (based on a literature review of CSE principles and recommended characteristics), implementation (assessing the execution of the program through the analysis of the reflection tools used by the educators), and short-term outcomes (assessing critical thinking and conscious behavioural choices through pre-post and satisfaction surveys). Results The main goal, learning modules and content were defined and structured in five interventions with the students, and two with families and teachers. A total of 638 students were involved in the activity, across 11 schools. Data analysis of pre/post surveys reported a significant increase in knowledge in 12 of the 15 items investigated ( p  < 0.05), and a high level of satisfaction with the topics addressed. Qualitative analysis added information on the pivotal role of educators in CSE. Conclusions The national piloting of this educational activity provided positive insights regarding the co-construction, implementation and short-outcome evaluation, suggesting potential for scalability and future inclusion of CSE in the curricula of Italian schools.

Aims Recent advances in Radiotherapy have led to the development of online adaptive RT (oART), a procedure addressing inter-fraction anatomical variations. Integrating artificial intelligence (AI) into the oART procedure speeds up the process and reduces user dependency. This study investigates the dosimetric advantage of implementing AI-driven oART in prostate cancer. Methods A total of 31 prostate cancer patients treated with oART on an AI-integrated Linac were analyzed. Patients were categorized by nodal involvement. For prostate-only cases, the Clinical Target Volume (CTV) included the prostate and seminal vesicles (CTV1), with a 5 mm margin (8 mm caudally) for Planning Target Volume (PTV), named PTV1. For nodal cases, pelvic lymph nodes were added (and categorized as CTV2) with a 5 mm isotropic margin (PTV2). Daily CBCTs were acquired, with OARs (rectum, bladder, bowels) automatically segmented by the AI system, while targets were manually delineated. Two plans were generated: a predicted one, calculating the original plan’s fluence on daily anatomy, and an adapted one, with complete fluence re-optimization. Daily DVH indicators for PTV(V95%), CTV(D98%), bladder (V65Gy), bowel (V45Gy), and rectum (V50Gy) were compared between predicted and adapted plans using the Wilcoxon-Mann-Whitney test. Total session time, from CBCT acquisition to treatment completion, was also recorded. Results oART treatment improved prostate coverage in both patient groups (+10.4% and +11.8% in PTV V95% for patients with and without lymph nodes) and CTV D98% (+2.6% with lymph nodes, +2.9% without). Improvements for arm 2 were smaller (+3.1% in PTV2 V95%, +2.2% in CTV2 D98%). Statistical differences were insignificant in OAR DVH indicators ( p  > 0.1). Median treatment time was 25 min and 32 min for prostate-only and lymph node cases, respectively. Conclusion This study demonstrates that oART in prostate cancer results in a significant improvement in target coverage with no significant difference in OARs.