Graphene is the most promising, versatile, and sustainable key enabling nanotechnology material which may bring solutions in the near future to many areas spanning materials science and medicine. However, the potential interactions of graphene with biological systems deriving from its chemico-physical properties and reactivity are raising public concern on risks for human health and environment. Independently from any specific purpose, a critical step for future translational applications is represented by the assessment of graphene impact on the immune system. Following any type of exposures, nanomaterials immediately contact the organism immune cells. Hence the investigation of the immune cell reactions is a milestone for the safe exploitation of graphene for future biomedical applications. G-IMMUNOMICS overall objective is to complement Flagship research on graphene safety with immunogenomic and proteomic data, not covered by the Core Project. G-IMMUNOMICS will provide new insights on the immune impact of several types of graphene to lay the basis for a safe use of graphene and future transfer in medicine. The specific objectives are to 1) Produce highly stable and dispersible pristine and functionalized graphene family materials (GFMs) with different lateral size and appropriate functionalizations 2) Characterize by high throughput functional immunogenomics and proteomics approaches and genotoxic assays the immune cell response induced by GFMs on different cell lines and primary cells 3) Evaluate the immune cell response induced by GFMs on four different species: human, mouse, pig and worm. G-IMMUNOMICS integrates with the Flagship at 3 levels: - Scientific. Well established collaborations are ongoing with many partners of the Flagship; in particular the project will synergize with the WP2 Flagship objectives. The project will: 1) extend the range of immune cell types investigated with a wide variety of populations and species; 2) implement the large scale OMICS research approaches assays (genomics and proteomics); 3) take part in the assessment of the environmental impact of the new materials by analysing the genotoxic effect on different organisms - Operational. The work plan builds on deep connections with the Flagship, due to: 1) the exploitation of synthesis protocol Flagship developed by WP1 and WP10; 2) the testing of GFMs provided by Flagship WP2 and WP10 partners; 3) the usage of human ex vivo and mouse in vivo samples for genomic, proteomic and genotoxic analysis provided by Flagship WP2. - Dissemination. The results gained during the project will be shared with Flagship partners during WP meetings and general assemblies in order to boost the transfer of knowledge achieved on graphene. Outcomes: G-IMMUNOMICS research may outline potential pathogenic biomarkers (genes, miRNA and proteins) associated with GFMs exposure, assess their overall safety profile and identify highly biocompatible GFMs suitable for future medical exploitation

Deregulation of cell cycle progression, associated with amplification, overexpression or hyperactivation of cell cycle regulators is one of the major hallmarks of cancer. In particular, it is well established that hyperactivation of cyclin-dependent kinases contributes to cancer cell proliferation in several human cancers. As such, and given their central role in coordination of cell division, as well as their prognostic value as indicators of poor disease outcome, these enzymes constitute attractive pharmacological targets for the development of cancer therapeutics. Unfortunately, diagnostic approaches for detecting alterations in the activities of these intracellular targets in a standardized fashion are poorly developed, involve indirect approaches such as immunohistochemistry, Reverse Transcriptase - Polymerase Chain Reaction, or mass spectrometry, and remain invasive, relying on biopsies, cell fixation or extraction procedures. We have recently developed several families of fluorescent peptide biosensors to probe the relative abundance of cyclin-dependent kinases, and to monitor their kinase activity, which have been validated in vitro using recombinant proteins and cell extracts and in living cells by molecular imaging. In the present program, we propose to develop multiplex sensing systems to probe different kinase activities simultaneously, which can be applied both ex vivo and in vivo, through functionalization of carbon nanotubes (CNTs) on the one hand, and through complexation with formulations of cell-penetrating peptide nanoparticles (CPPs) , on the other hand, with fluorescent peptide biosensors. The use of CPPs will allow to prepare non-covalent complexes with several different biosensors, while the CNTs will be used as a platform to prepare covalent complexes. Aside from their attractivity for multifunctionalization, one of the most attractive features of CNTs is their ability to cross cell membranes through a mechanism that is reminiscent of a “nanoneedle”. Likewise CPPs constitute efficient carriers for intracellular delivery of a variety of different biomolecules, including peptides bearing synthetic fluorescent probes. The physico-chemical features of biosensor-nanotubes and of biosensor-nanoparticles will be characterized, and their ability to detect different kinase activities will be validated in vitro with recombinant kinases and cell extracts. These fluorescent “nanomultisensors” will then be introduced into living cells, and their ability to report on different kinase activities will be investigated in different physiological and pathological settings to assess their specificity and sensitivity. Moreover, the potential inhibitory effect and cytotoxicity of these nanosystems on cell viability and proliferation will be characterized carefully. Nanotube and nanoparticle multisensors will then be introduced into animal models through injection, and their biodistribution, as well as their ability to report on pathological alterations of cyclin-dependent kinases in xenografted cancer cells, prior to and following administration of therapeutics, will be assessed through multispectral imaging. NANOMULTISENS program will have a significant impact in life sciences and nanotechnology, in terms of medical and technological applications. Technological innovations will include the engineering of functionalized carbon nanotubes and peptide-based nanoparticles for simultaneous delivery of different fluorescent biosensors in living cells and in vivo, and their application for multiplex sensing of different kinase activities through molecular imaging. These fluorescent nanomultisensors will enable development of smart and personalized diagnostics, by contributing to establish strategies for early detection of alterations associated with cancer cell proliferation, for monitoring disease progression and response to therapeutics.

Lymph nodes (LN) are prototypical lymphoid organs where adaptive immune responses against local infections are initiated. It has become well established that peripheral dendritic cells (DC) arriving from afferent lymphatic vessels in the subcapsular sinus (SCS) migrate via the interfollicular areas (IFA) into the paracortical T cell zone. There, DC present processed protein antigen in the form of peptide-major histocompatibility complex (pMHC) to naive T cells for the initiation of cellular responses. This process has been exhaustively studied. Yet, IFA are strongly enriched for cells of the innate immune system such as gamma-delta T cells, innate-like CD8+ T cells, natural killer T cells (NKT), innate lymphoid cells (ILC), natural killer (NK) cells, macrophages and neutrophils. The physiological function of this multicellular innate system has been assigned to limit pathogen spread by fostering antimicrobial activity of macrophages and stromal cell-mediated repair processes. To date, however, it is unknown how the specific IFA niche for innate immune cells is created and maintained by the stromal microenvironment. Further, it remains unclear whether and how a microbial challenge impacts on innate cells of the IFA-resident multicellular immune system and their cross-talk with cells of the adaptive immune system. The team of C. Mueller, partner in this project, has recently demonstrated that LN stromal mesenchymal cells located just beneath the SCS express the TNF family member TNFSF11 (also known as RANKL) to activate the overlaying TNFRSF11a+ (RANK+) lymphatic endothelial cells (LEC). This creates a specific niche for the recruitment and retention of sinusoidal macrophage (SM) populations. Deletion of mesenchymal RANKL or lymphatic RANK precipitates the loss of SM, with a concomitant impairment of humoral immune responses. In addition, LEC lose their activated phenotype, which leads to altered expression of numerous immunoregulatory genes. These include chemokine scavenging receptors and the chemoattractants CXCL4 and CCL20, which are known to attract and/or retain cells of the innate immune system. An attractive hypothesis is therefore that the RANKL-RANK signaling axis linking LN mesenchymal cells and LEC orchestrates the establishment and maintenance of the multicellular innate immune system of the IFA. Here, we will combine transgenic mouse lines with disrupted RANK and CSF-1 signaling in combination with reporter mouse lines for innate immune cells to critically examine the role of LEC in establishing the innate immune system in IFA. To this end, we will employ multiplex immunofluorescence in combination with whole-organ and intravital imaging, flow cytometry, as well as single cell and spatial transcriptomics of the IFA niche in the steady state and after microbial challenge. We will use this knowledge to examine how activated LEC and the innate immune system of the IFA regulate adaptive immunity during a microbial challenge. From these data, we anticipate to unravel the complex cross-talk balancing innate and adaptive immune responses in reactive LN, which may eventually lead to specifically-tailored vaccination strategies.

Tissue-resident macrophages (TMs), including epidermal Langerhans cells (LCs), are long-lived cells able to proliferate. Their ontogeny and adaptation to different organs have been studied in great details, yet their physiological maintenance deserves further investigations. Autophagy, a catabolic process regulated by autophagy-related (Atg) genes, prevents accumulation of harmful cytoplasmic components and mobilizes energetic reserves in long-lived and self-renewing cells. Recently, we found that Atg5-deficient LCs undergo apoptosis as a result of lipid metabolism dysregulation. Here, we propose that autophagy allows TMs to manage lipid stocks and ensure long-term maintenance. We will first validate this in murine and human LCs, then verify whether it holds true for TMs of the lung, liver and lymph nodes. Finally, we will test whether autophagy could permit TMs to adapt to cellular stress and limit inflammation induced by metabolic alterations, irradiation, aging and viral infection. Altogether, our results will introduce a new paradigm on the maintenance of TMs in a broad range of organs under physiological and inflammatory conditions.

Graphene-based materials (GBMs), including graphene (G) and graphene oxide (GO), start to be present in all environmental compartments i.e. air, water and soils as a consequence of their diverse industrial developments. Thus, it is of fundamental interest to question about the potential adverse effects of these materials on organisms as well as their persistence in the environment. Despite the abundant bibliography focused on GBMs, there is still a limited number of studies reporting in vivo biological degradation of this type of two-dimensional nanomaterials. The aim of this proposal is to combine the knowledge and expertise of different research groups in microbiology, biotechnology and nanomaterials to explore the ability of environmental bacteria to degrade and to interact with G and GO. To perform the project, a straightforward approach will be used. First, samples from graphitic deposit sites will be collected and the diversity of indigenous bacterial communities will be deciphered and identified. Culture-dependent methods with diverse growth media and abiotic conditions (T, pH) for enriched cultures will be implemented, along complementary culture-independent methods. 16S rDNA gene sequencing of extracted DNA will be used to infer taxonomic identifications. Second, as naphthalene degraders would be also potential GBM degraders, screening of collections of bacteria (belonging to the partners of this project) isolated from hydrocarbons and metals contaminated sites for this function will be performed. Then, experiments will be set up in continuous flow-cell and columns reactors, and in batch and semi-batch flask reactors, in which fully characterized GBM materials could interact with the previously isolated bacteria, as a single strain or bacterial consortia during 45 days. The kinetic monitoring of G and GO degradation/modifications by the selected bacteria will be investigated at regular intervals by electron microscopy (TEM/SEM) and spectroscopy (XPS, Raman) methods. Besides, experiments with 13C-labelled GBMs will allow us to determine by HR-MAS NMR the carbon uptake/assimilation by bacteria. Finally, interactions of bacteria with G and GO leading to degradation of these materials or to surface modifications will also be investigated in bioflotation experiments as they can provide insights on the degradation mechanisms. Thus, the project targets to isolate bacteria, from environmental graphite-containing samples and/or strain collections, which possess potent abilities towards the degradation of GBMs. Achieving this objective, the project will improve, refine and fill the gaps of the current knowledge on the environmental fate of GBMs and more particularly on the bacterial contribution to the bioprocesses of degradation. The findings of this basic research project will help to develop prospective bioremediation processes as well as to resolve a soon-to-be societal issue.