IRCCS San Raffaele Scientific Institute Showcase

During embryonic development, blood cells emerge from specialized endothelial cells, named hemogenic endothelial cells (HECs). As HECs are rare and transiently found during very early development, it remains difficult to distinguish them from endothelial cells. Here, we performed transcriptomic analysis of 28-32 day human embryos, and observed that the expression of Fc receptor CD32 (FCGR2B), is highly enriched in the endothelial cell population that contains HECs. Functional analyses using human embryonic and hPSC-derived endothelial cells revealed that robust multilineage hematopoietic potential is harbored within CD32+ endothelial cells and showed that 90% of CD32+ endothelial cells are bona fide HECs. Remarkably, these analyses indicated that HECs progress through different states, culminating in FCGR2B expression, at which point cells are irreversibly committed to a hematopoietic fate. These findings provide a precise method for isolating HECs from human embryos and hPSC cultures, thus allowing the efficient generation of hematopoietic cells in vitro.

In physiological conditions, few circulating hematopoietic stem/progenitor cells (cHSPC) are present in the peripheral blood but their contribution to hematopoietic homeostasis in humans remain unsolved. By integrating advanced immunophenotyping, cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq), functional single-cell assays and integration site (IS) clonal tracking, we unveiled the phenotypic composition, the transcriptional features and the biological role of human cHSPC subpopulations in relationship to their bone marrow (BM) counterpart. We found that cHSPC progressively reduced in cell count over aging and are enriched for primitive, lymphoid and erythroid subpopulations, showing pre-activated transcriptional and functional state. Moreover, cHSPC have low expression of multiple BM-retention molecules, but maintain their homing potential after xenotransplantation. By generating a comprehensive Human Organ-Resident HSPC (HuOR) dataset based on scRNAseq data, we detected organ-specific seeding properties of the distinct trafficking HSPC subpopulations. Of note, circulating multi-lymphoid progenitors (MLP) are primed for seeding the thymus and actively contribute to T-cell production at steady state in patients treated with HSPC-gene therapy (GT). Human clonal tracking data from GT patients also showed that cHSPC connect distant BM niches and participate to steady-state hematopoietic production, with primitive cHSPC having the highest re-circulation capability to travel in and out the BM. Finally, in case of hematopoietic impairment, cHSPC composition reflects the BM-HSPC content and might represent a biomarker of the BM state for clinical and research purposes. Overall, our comprehensive work unveiled fundamental insights into the in vivo dynamics of human HSPC trafficking and its role in sustaining hematopoietic homeostasis.

Plasma membrane-associated platforms (PMAPs) form at specific sites of plasma membrane by scaffolds including ERC1 and Liprin-1. We identify a mechanism regulating PMAPs assembly, with consequences on motility/invasion. Silencing Ser/Thr kinase DYRK3 in invasive breast cancer cells inhibits their motility and invasive capacity. Similar effects on motility were observed by increasing DYRK3 levels, while kinase-dead DYRK3 had limited effects. DYRK3 overexpression inhibits PMAPs formation nad has negative effects on stability of lamellipodia and adhesions in migrating cells. Liprin-1 depletion results in unstable lamellipodia and impaired cell motility. DYRK3 causes increased Liprin-1 phosphorylation. Increasing levels of Liprin-1 rescue the inhibitory effects of DYRK3 on cell spreading, suggesting that an equilibrium between Liprin-1 and DYRK3 levels is required for lamellipodia stability and tumor cell motility. Our results show that DYRK3 is relevant to tumor cell motility, and identify a PMAP target of the kinase, highlighting a new mechanism regulating cell edge dynamics. Upon request, the corresponding author is available to provide the data in different formats if it is not possible to open data files with open-source softwares.

In the manuscript "IL-10-producing regulatory cells impact on celiac disease evolution" (doi: 10.1016/j.clim.2024.109923), we showed that IL-10-producing cells are fundamental in controlling pathological T-cell responses to gluten. In brief, our major findings are: 1) Gluten ingestion induces systemic inflammation in CD patients; 2) Gliadin-specific IFN-g+ T cells are present in patients regardless of tissue damage; 3) The presence of DC-10 in mucosal infiltrates is a hallmark of potential CD patients; 4) In potential CD, DC-10 maintain mucosal homeostasis and protect the gut from damage. The datasets here loaded contain all the clinical and immunological data, supporting our conclusions, that have been used to build the manuscript. An additional file (raw_data_description) contains the main guidelines to correctly associate the raw data with the corresponding figure/table in the manuscript.

Raw data associated to the paper Zanardi, Nardini, et al. Communications Biology (see link). - Proteomics raw data for protein identification associated to Figure 1 of the manuscript are deposited in dedicated repository indicated in the paper (see "link") - Folder " 64CUkCP Biodistribution ": raw data associated to Fig 5 panel (a) and Supplementary Figure 8 describing labelled Cp biodistribution in the brain and blood of mice - Folder "Images choroid plexus": collection of the histochemistry images of choroid plexus stained for iron deposition used to generate data of figure 5 panels (g and h) - Folder "Images Purkinje": collection of the histochemistry images of cerbellum used to count thenumber of Purkinje neurons reported on figure 5 panels (i and j) - Folder "Images liver": collection of the histochemistry images of liver stained with hematoxylin-eosin used to generate figure 7 panels (c and d) - Folder Immunohistochemistry images Cerebellum => Autofluor Purkinje Cells Density: images used to generate data in figure 5 panels (k and l) - Folder Immunohistochemistry images Cerebellum => Astrocyte Density in Arbor Vitae: images used to generate data in figure 6 panels (a and b) - Folder Immunohistochemistry images Cerebellum => GFAP expression in Arbor Vitae: images used to generate data in figure 6 panel (c) - Folder Immunohistochemistry images Cerebellum => Microglia Density in Arbor Vitae: images used to generate data in figure 6 panels (a and d) - Folder Immunohistochemistry images Cerebellum => IBA1 expression in Arbor Vitae: images used to generate data in figure 6 panel (e) - Folder Immunohistochemistry images Hippocampus => Astrocyte Density in CA1 SR: images used to generate data in figure 6 panels (f and g) - Folder Immunohistochemistry images Hippocampus => GFAP expression in CA1 SR: images used to generate data in figure 6 panel (h) - Folder Immunohistochemistry images Hippocampus => Microglia Density in CA1 SR: images used to generate data in figure 6 panels (i and j) - Folder Immunohistochemistry images Hippocampus => IBA1 expression in CA1 SR: images used to generate data in figure 6 panel (k) - Folder Immunohistochemistry images Hippocampus => Neuron Density in CA1 SP: images used to generate data in figure 6 panels (l and m) - Folder Immunohistochemistry images Hippocampus => Thickness of CA1 SP: images used to generate data in figure 6 panel (n) - Folder Immunohistochemistry images Hippocampus => Autofluor Neurons Density in CA1 SP: images used to generate data in figure 6 panel (o) - Folder "WT+CP database ": raw data associated to Supplementary Figure 6 describing the evaluation of toxicity induction in wild-type (WT) mice treated for 4 months with purified ceruloplasmin

- Raw images of liver histological sections stained either with hematoxylin-eosin or with anti-F4/80 antigen antibody (counterstaining with hematoxylin) - Raw images of adipose tissue histological sections stained either with hematoxylin-eosin or with anti-F4/80 antigen antibody (counterstaining with hematoxylin) - Raw data from MRS and NMR spectra (files can be used with MestreNova software, Mestrelab Research, S.L. Santiago de Compostela, Spain)

Recombination activating genes (RAG) 1 and 2 defects are the most frequent form of severe combined immunodeficiency (SCID). Patients with residual RAG activity have a spectrum of clinical manifestations ranging from Omenn syndrome to delayed-onset combined immunodeficiency, often associated with granulomas and/or autoimmunity (CID-G/AI). Lentiviral vector (LV) gene therapy (GT) has been proposed as an alternative treatment to the standard hematopoietic stem cell transplant and a clinical trial for RAG1 SCID patients recently started. However, GT in patients with hypomorphic RAG mutations poses additional risks, because of the residual endogenous RAG1 expression and the general state of immune dysregulation and associated inflammation. In this study, we assessed the efficacy of GT in 2 hypomorphic Rag1 murine models (Rag1F971L/F971L and Rag1R972Q/R972Q), exploiting the same LV used in the clinical trial encoding RAG1 under control of the MND promoter. Starting 6 weeks after transplant, GT-treated mice showed a decrease in proportion of myeloid cells and a concomitant increase of B, T and total white blood cells. However, counts remained lower than in mice transplanted with WT Lin- cells. At euthanasia, we observed a general redistribution of immune subsets in tissues, with the appearance of mature recirculating B cells in the bone marrow. In the thymus, we demonstrated correction of the block at double negative stage, with a modest improvement in the cortical/medullary ratio. Analysis of antigen-specific IgM and IgG serum levels after in vivo challenge showed an amelioration of antibody responses, suggesting that the partial immune correction could confer a clinical benefit. Notably, no overt signs of autoimmunity were detected, with B-cell activating factor decreasing to normal levels and autoantibodies remaining stable after GT. On the other hand, thymic enlargement was frequently observed, although not due to vector integration and insertional mutagenesis. In conclusion, our work shows that GT could partially alleviate the combined immunodeficiency of hypomorphic RAG1 patients and that extensive efficacy and safety studies with alternative models are required before commencing RAG gene therapy in these highly complex patients.

Raw data and original images for the article entitled "Boosting Interleukin-12 Antitumor Activity and Synergism with Immunotherapy by Targeted Delivery with isoDGR-Tagged Nanogold" by Gasparri AM, Sacchi A, Basso V, Cortesi F, Freschi M, Rrapaj E, Bellone M, Casorati G, Dellabona P, Mondino A, Corti A, Curnis F. Small. 2019 Nov;15(45):e1903462. doi: 10.1002/smll.201903462.

TDC are hematopoietic cells that combine dendritic cell (DC) and conventional T cell markers and functional properties. They were identified in secondary lymphoid organs (SLOs) of naïve mice as cells expressing CD11c, major histocompatibility molecule (MHC)-II, and the T cell receptor (TCR)  chain. Despite thorough characterization as to their potential functional properties, a physiological role for TDC remains to be determined. Unfortunately, using CD11c as a marker for TDC has the caveat of its upregulation on different cells, including T cells, upon activation. Therefore, a more specific marker is needed to further investigate TDC functions in peripheral organs in different pathological settings. Here we took advantage of Zbtb46-GFP reporter mice to explore the frequency and localization of TDC in peripheral tissues at steady state and upon viral infection. RNA sequencing analysis confirmed that TDC identified with this reporter model have a gene signature that is distinct from conventional T cells and DC. In addition, frequency and total numbers of TDC in the SLOs recapitulated those found using CD11c as a marker. This reporter model allowed for identification of TDC in situ not only in SLOs but also in the liver and lung of naïve mice. Interestingly, we found that TDC numbers in the SLOs increased upon viral infection, suggesting that TDC might play a role during viral infections. In conclusion, we propose a visualization strategy that might shed light on the physiological role of TDC in several pathological contexts, including infection and cancer.

Raw data and original images for the article entitled "NGR-TNF Engineering with an N-Terminal Serine Reduces Degradation and Post-Translational Modifications and Improves Its Tumor-Targeting Activity" by Angelo Corti, Anna Maria Gasparri, Angelina Sacchi, Barbara Colombo, Matteo Monieri, Eltjona Rrapaj, Andrés J. M. Ferreri, and Flavio Curnis Molecular Pharmaceutics 2020 17 (10), 3813-3824, DOI: 10.1021/acs.molpharmaceut.0c00579