Controlling virus replication by generating a strong CD8+ T cell response against HIV is one of the major goals in the development of an effective HIV vaccine candidate. Indeed, during the chronic phase of infection, HIV-specific CD8+ T cells were shown to have impaired functionality and fail to control viral replication. Understanding the profile of CD8+ T cells able to efficiently tackle HIV is therefore much needed. HIV controllers (HIC) are individuals who control viremia without antiretroviral therapy. CD8+ T cells seems to play a major role in their HIV control. We hypothesized that HIV-specific CD8+ T cells associated with control of infection bear particular transcriptional signature (cell cycle, survival, activation, cytolytic function…) when compared to HIV-specific CD8+ T cells not associated with control of infection sharing the same memory phenotype.

La PF Transcriptome et Epigenome développe des projets de séquençage à haut débit (collaboration et service) avec des équipes du Campus. Ceux-ci couvrent l'ensemble des thématiques du campus ainsi qu'une large gamme d'organismes (des virus aux mammifères). La plate-forme exerce des activités de biologie humide (construction des librairies et séquençage) et de biologie sèche (analyse bioinformatiques et statistiques). La personne mise a disposition interagira étroitement avec les autres bioinformaticiens du pôle BioMics et du Hub. Ses activités concerneront notamment: - La participation à la conception et à la mise en place des projets avec les équipes demandeuses, la prise en charge des analyses et le reporting aux utilisateurs - La mise en place d'un workflow d'analyse bioinformatique des données de transcriptome /épigénome en étroite collaboration avec le C3BI, la DSI et les autres bioinformaticiens du pole. Ce workflow permettra le contrôle qualité des données, leur prétraitement, le mapping des séquences sur les génomes/transcriptomes de réference, et le comptage des reads pour les différents éléments de l'annotation - L'adaptation du workflow d'analyse aux questions biologiques et aux organismes étudiés dans le cadre des activités de la PF - L'activité de veille technologique et bibliographique (test et validation de nouveaux outils d'analyse, updates d'outils existants...) - La mise en place et le développement d'outils d'analyse adaptés aux futurs projets de la PF: single cell RNAseq, métatranscriptome, ChIPseq, analyse des isoformes de splicing.. Ceci se fera notamment via la réalisation d'analyses dédiées avec certains utilisateurs. Les outils mis en place et validés dans ce cadre seront ensuite utilisés pour l'ensemble des projets. - L'activité de communication et de formation (participation aux réunions du consortium France Génomique,formation permanente à l' Institut Pasteur… - la participation a d autres projets du Pole BioMics (selon disponibilité) Bernd Jagla, qui était le bioinformaticien de la plateforme a rejoint le Hub au 1er janvier 2016. Rachel Legendre est mise a disposition depuis le 2 novembre 2015 et remplace Bernd Jagla. Je souhaite que Rachel Legendre soit mise à disposition de la plateforme pour une durée d'au moins 2 ans.

The Center for Human Immunology (CIH) supports researchers involved in translational research projects by providing access to 16 different cutting edge technologies. Currently, the CIH hosts over 60 scientific projects coming from 8 departments of the Institut Pastuer and 5 external teams. In order to respond to the growing needs of these projects in the area of single cell analysis, the CIH has introduced a significant number of single-cell/single-molecule technologies over the past 2-3 years. These new technologies, such as the Personal Genome Machine (PGM) and Ion Proton sequencers, iSCAN microarray scanner, Nanostring technology for transcriptomics profiling and real-time PCR machine BioMark, give rise to large datasets with high dimensionality. Such trend, in terms of data complexity, is also true for flow cytometry technologies (currently reaching over 20 parameters per cell). The exploration of this data is generally beyond the scope of scientists involved in translational research projects. In order to maximize the research outcomes obtained from the analysis of these rich datasets, and to ensure that the full potential of our technologies can be served to the users of the CIH, we would require a proximity bioinformatics support. A CIH-embedded bioinformatician would: 1) design and implement standard analysis pipelines for each of the data-rich technologies of the CIH; 2) provide regular ‘bioinformatics clinics’ to allow scientists the possibility to customize standard pipelines to their specific needs; 3) run trainings on the ‘R software’ platform and other data analysis tools (such as Qlucore) of interest for the CIH users. The objective would be to empower the users to run exploratory analysis by themselves, and to teach good practices in terms of data management and data analysis.    

The Transcriptome & Epigenome Platform is dedicated to the development and use of high throughput approaches for transcriptomics and epigenomics studies. The platform is accessible to any research team from the Pasteur Institute (80% of the projects) as well as from outside. It is involved (most often as collaborator) in several projects funded by the ANR, Microbes and Brain, ERANET and by the Pasteur Institute in the framework of the PTR programs. Next Generation Sequencing (NGS) based on the Illumina technology (HiSeq 2000/2500 sequencers) is used to perform RNA-sequencing experiments for which a large amount of data is generated. After a first step involving bioinformatics, specific statistical methods must be used be analyze rigorously the data. These analyses are most often performed by the statistician(s) of the platform. They are also in charge of bibliographical survey activity.

Detection of nucleic acids at the single cell level using microscopy has now reach high throughput levels which promise exciting discoveries concerning the functionning of the genomes. The HSC3D project funded by the CITECH in the Pasteur institute aims at multiplexing nucleic acid detection by Fluorescence In Situ Hybridisation (FISH). This necessitates libraries in the range of tens of thousands of  100mer DNA oligonucleotides which will be synthesised by digital lithography. Several constrains apply to these oligonucleotides which therefore require heavy duty bioinformatics for their design .

Over the last years, innate lymphoid cells (ILC) have been increasingly investigated. Despite the absence of antigen specific receptors, they belong to the lymphoid lineage and represent important sentinels for tissue homeostasis and inflammation. They contribute to numerous homeostatic and pathophysiological situations via specific cytokine production. ILC are currently divided into three groups based on the expression of specific transcription factors and secretion of cytokines. We focus this study on fetal ILC3 development. We have observed that contrary to lymphocytes, ILC can migrate toward lymphoid organs, tissues and mucosal sites as lymphoid precusors and terminate their developmental program in situ. In the fetal spleen, we observe different stages of ILC3 with precursors that are already RORgt+ but could still give rise to other ILC fate. Hence, these splenic ILC3 precursors were sorted and analyzed by microarrays. The identification of gene expression differences was used to design a single cell transcriptomic assay. The single cell transcriptomic assay is based on this specific selection of primers for transcription factors and cytokine receptors. We evaluate their differential expression in single cells at different stages of their plasticity. The aim is to decipher the progression from an ILC precursor stage to an another in one cell. We are also using the new polaris technology to detect and evaluate at different early timepoints the sequence of molecular events for changing ILC cell fate. In this case, we chose to use the sc RNAseq technology. The single cell transcriptomic will be analyzed and bioinformatic programs will be applied in order to organize the sequential molecular events and to build a hierarchical developmental model in case of ILC cell fate decisions.

The rare patients who spontaneously control HIV replication in the absence of therapy show signs of a particularly efficient cellular immune response. To identify the molecular determinants underlying this response, we characterized the TCR repertoire directed at the most immunodominant CD4 epitope in HIV-1 capsid, Gag293. HIV Controllers from the ANRS CO21 CODEX cohort showed a highly skewed TCR repertoire characterized by a predominance of the TRAV24 and TRBV2 variable gene families. Controllers shared public clonotypes at higher frequencies than treated patients, suggesting the implication of particular TCRs in HIV control (Benati D. et al., J Clin Invest 2016).   We propose to test the generality of these findings by characterizing the TCRs specific for a series of immunodominant HIV Gag and Env epitopes, and comparing the frequencies of public clonotypes in groups of HIV Controllers and treated patients. We will then assay the functions of the most prevalent public clonotypes through lentivector-based TCR transfer, and correlate the panel of T cell functions to TCR affinity and frequency.

HIV infects and depletes CD4+ T cells, leading to a progressive loss of adaptive immune responses and ultimately to AIDS. In addition, HIV preferentially targets HIV-specific CD4+ T cells, resulting in an attrition of the very cells that should orchestrate the antiviral immune response. Due to this preferential depletion, HIV-specific CD4+ T cells are few and remain incompletely characterized. The emergence of single cell technologies opens the opportunity for an in depth analysis of the rare specific CD4+ T cells that persist in the circulation of chronically infected patients. We have sorted single CD4+ T cells specific for the most immunodominant epitope in HIV-1 capsid, using an optimized MHC class II tetramer labeling protocol. We now propose to analyze these single cells by multiplexed real time PCR in a microfluidics device, to define their transcriptional profile. We will analyze the differentiation status of HIV-specific CD4+ T cells in rare patients who naturally control HIV infection and in progressor patients who control HIV replication due to antiretroviral therapy. This project will help define the parameters of an efficient T cell response against HIV, and may provide insights into the type of responses that should be induced by candidate HIV vaccines.

Quantitatively understanding the stochastic dynamics of gene expression requires measurements at the level of single cells. A common approach to follow the expression of genes in single cells and in real time is to make use of fluorescent reporter proteins and to record the cells' fluorescence by microscopy. However, this provides only an indirect readout of the biological processes that are of interest such as the regulation mechanisms at the promoter. A possible way to uncover the unobservable biological processes is to infer the hidden dynamics from the available data through the use of mechanistic models of gene expression. The goal of this project is to develop methods for state estimation and parameter inference for such models and to test these methods on real data.

Invasion of human intestinal epithelial cells by Shigella flexneri is secondary to the delivery of bacterial effectors into the host cell cytoplasm via a type III secretion system (T3SS). By using a beta-lactamase reporter tool we observed that in contrast to the epithelium, human lymphocytes are mainly targeted by injection of T3SS effectors not resulting in subsequent cell invasion (Pinaud et al., 2017). Furthermore, we observed that the targeting process, in form of successful injection of effectors into the host cell, is dependent on glycan-glycan interactions between bacterial and host cell surfaces rendering the targeting process to be dependent on the activation state of the host cell (Belotserkovsky et al., 2018). CyTOF technology is a research tool used for phenotypic analysis of complex cell population allowing for the simultaneous labelling of up to 40 different surface and intracellular marker without issues of compensation as present in regular flow cytometry (van Unen et al., 2016). Using CyTOF technology and the beta-lactamase reporter tool, we will perform a detailed analysis of Shigella targeting in a complex cell population using human lamina propria mononuclear cells (LPMCs), isolated from human colon explants. Analysis will address the question if specific cellular subsets are preferentially targeted in the intestinal environment and if this differs from targeting of peripheral blood mononuclear cells (PBMCs) diverging in their immune phenotypes and cellular activation.

Erythromyeloid progenitors (EMPs) originate from the yolk sac during early mouse development and migrate to the fetal liver via the circulation where they undergo massive expansion and differentiation into hematopoietic lineages. These events occur prior to the intraembryonic emergence of hematopoietic stem cells (HSCs). Unlike HSCs, EMPs cannot give rise to lymphoid lineages, nor can they provide long-term repopulation. As such, they are considered a transient fetal population, yet it is EMP-derived hematopoiesis that supports the growth and survival of the embryo prior to the establishment of long-term hematopoitic stem cells (HSCs). Hematopoietic cell differentiation occurs along a hierarchy of progenitors with either lymphoid or myeloid fates. Common myeloid progenitors (CMPs) give rise to further restricted granulocyte-monocyte progenitors (GMPs) and megakaryocyte-erythrocyte progenitors (MEPs). This hierarchy has been well documented in adult hematopoiesis, which occurs solely from HSCs. However, fetal hematopoiesis encompasses dual origins of myeloid lineages that can originate from either EMPs or HSCs. Using genetic pulse chase labeling, we are able to distinguish these two ontologies by positively labelling EMPs and their progeny. Currently, fetal liver progenitors have been characterised by direct comparison to markers and expression profiles that are established for adult hematopoiesis. Yet, fetal hematopoietic markers may not be regulated in the same manner as their adult counterparts. Furthermore, distinguishing EMP- versus HSC-derived progeny is technically challenging and has not been properly addressed with respect to fetal liver myelopoiesis. Therefore, using our genetic pulse chase labeling approach, we would like to rebuild the differentiation tree among myeloid fetal liver progenitors. We are using high parameter flow cytometry to re-evaluate progenitor sub-populations with an expanded repertoire of markers. Since heterogeneity among progenitors (in terms of gene expression and differentiation potential) can be misrepresented and difficult to characterize on the population level, we want to investigate this on the single cell level using MARS-Seq in combination with index sorting.

Tissue resident stromal cells form the scaffold of all organs. In addition, they provide signals for proper positioning, survival and interaction of a number of other cell types, such as immune cells. Following tissue damage, the stromal microenvironment play an esssential role to organize inflammation and tissue repair. The stromal vascular fraction (SVF) of most organs is composed of immune cells, endothelial cells, mesenchymal stromal cells and tissue stem cells. Our lab investigates the impact of specific subsets of mesenchymal stromal cells (MSCs) on the different components of the SVF, at homeostasis and during the repair process. We have previoulsy identified specific subsets of MSCs that have an essential role in inflammation, tissue fibrosis or maintenance of the intestinal stem cell niche (Peduto et al., 2009; Dulauroy et al., 2012; Stzepourginski et al., 2017). This project aims at further deciphering the heterogeneity of the stromal compartment in different organs at homeostasis or following injury, and establish its transcriptional signature.

The development of the mammary gland occurs in five distinct phases: embryogenesis, puberty, pregnancy, lactation, and involution. Due to its extraordinary regenerative capacity, the mammary epithelium is a fantastic system to study the physiological regulation of cellular plasticity in vivo. It comprises two major cellular lineages, the outer myoepithelial (also called basal) and inner luminal cell layers. Although the existence of post-natal bipotent mammary stem cells (MaSCs) remains debatable, it is well accepted that there are subtypes of epithelial cells (progenitors, unipotent and/or bipotent stem cells) that are responsible for the remodeling and renewal of the mammary gland. Moreover, these cells have also been suggested to be the cell of breast cancer origin. Therefore, it is crucial to further understand how mammary gland maintains its proper cellular plasticity, especially during the cycles of pregnancy and involution, which might shed new lights on the initiation, progression, and metastasis of breast cancer.

Stromal cells are essential during organ morphogenesis and for the maintenance of tissue homeostasis. In addition, increasing evidence indicates that stromal cells play a role in certain type of chronic diseases, such as cancer. In this project, we will investigate stromal cell heterogeneity at the single cell level in specific organs at homeostasis and during pathology.

Erythromyeloid progenitors (EMPs) originate from the yolk sac during early mouse development and migrate to the fetal liver via the circulation where they undergo massive expansion and differentiation into hematopoietic lineages. These events occur prior to the intraembryonic emergence of hematopoietic stem cells (HSCs). Unlike HSCs, EMPs cannot give rise to lymphoid lineages, nor can they provide long-term repopulation. As such, they are considered a transient fetal population, yet it is EMP-derived hematopoiesis that supports the growth and survival of the embryo independently of HSCs. HSC differentiation respects a hierarchy of progenitors with either lymphoid or myeloid fates and has been well documented in adult hematopoiesis. However, fetal hematopoiesis ensues from erythroid and myeloid progenitors with dual origins from either EMPs or HSCs. Using a genetic pulse chase labeling approach to distinguish these two ontogenies, we would like to explore heterogeneity on the single cell level and to build the differentiation tree among fetal liver myeloid progenitors using MARS-Seq.

This projects aims at characterizing at the single cell level the stromal microenvironment of the skeletal muscle, to get a better insight into the heterogeneity and function of different cell populations involved in skeletal muscle homeostasis and regeneration.

Gene-modified T cells expressing a chimeric antigen receptor (CAR) targeting the CD19 molecule have demonstrated promising clinical efficacy in the treatment of B cell malignancies. However, the frequent relapses and toxic adverse events such as cytokine release syndrome represent hurdles to the success of CAR T cell therapies. In most clinical settings, CAR T cells are generated from a mixture of autologous CD4+ and CD8+ T cells before being infused into patients. This inter-patient heterogeneity within the composition of CAR T cell products renders the large variety of response efficacy and toxicity difficult to interpret. Using a model of B cell aggressive lymphoma developing in the bone marrow, we investigate the differential contributions of CD4+ and CD8+ anti-CD19 CAR T cells to tumor outcome and changes in the tumor microenvironment. Our first in vivo imaging and flow cytometry results suggest that CD4+ CAR T cells have poor cytotoxic potential compared to CD8+ CAR T cell. On the other hand, CD4+ CAR T cells were largely responsible for the cytokine release syndrome and have a unique role in boosting the accumulation of NK cells at the tumor site. Using single cell RNAseq, we aim to identify the changes in the bone marrow tumor microenvironment induced by CD4+ and CD8+ CAR T cells, focusing on the recruitment of host specific immune cell populations and their activation status. Identifying specific contributions of the CD4+ and CD8+ CAR T cells to immune cell recruitment and tumor outcome would help designing optimal CAR T cell products, with important clinical implications.

The global aim of the project is to characterize and explore the role of dermal macrophage populations in the control of vascular integrity in health and diseases. Currently, we are limited by the lack of population-specific markers that would allow us to study them down to the molecular level. We thus propose to perform a single cell gene expression analysis on pan leukocytes (CD45+) isolated from mouse skin dermis and epidermis in order to identify and define the genetic signature of the steady-state dermal macrophage subpopulations.

The rare patients who spontaneously control HIV replication in the absence of therapy show signs of a particularly efficient T cell response. We aim at characterizing the molecular determinants underlying this efficient antiviral response. We have previously shown that CD4+ T cells from HIV controllers express T cell receptors (TCRs) of particularly high affinity for Gag peptides/ MHC II complexes. Furthermore, HIV controllers shared Gag-specific TCR clonotypes at higher frequencies than treated patients, suggesting the implication of particular TCRs in HIV control. To extend these studies, we propose to characterize the TCR repertoire and the transcriptional pattern of HIV-specific CD4+ T cells at the single cell level. We will compare HIV-specific CD4+ T cells obtained from HIV controllers included in the ANRS CO21 CODEX cohort to those from patients receiving antiretroviral therapy.

The enteropathogen, Shigella, is highly virulent and remarkably adjusted to the intestinal environment of its almost exclusive human host. Key for Shigella pathogenicity is the injection of virulence effectors into the host cell, via its type three secretion system (T3SS), initiating disease onset and progression. T3SS-mediated host cell targeting is associated with Shigella invasion and dissemination in epithelial cells. Yet, our group reported the direct targeting of human lymphocytes by an injection-only mechanisms, i.e. the injection of Shigella T3SS effectors without subsequent cell invasion. To further investigate the scope and selectivity of this targeting mechanism, we utilised human lamina propria mononuclear cells (LPMCs) isolated from colonic explants and peripheral blood mononuclear cells (PBMCs) and assessed the mode of Shigella targeting by the use of a T3SS reporter stain and multiparametric CyTOF technology. In human LPMCs, a population of CD38hi plasma cells/ plasmablasts was identified as primarily targeted and presented up to 80% of all targeted LPMCs. Interestingly, plasma cells constitute the primary IgA-producing cell type and IgA has been previously identified as important mediator of Shigella pathogenicity. The functional implications on plasma cell targeting by Shigella is subject of current investigations.

Left-right asymmetry of the heart is essential for establishing the double blood circulation. Impairment of left-right embryo patterning is associated with severe congenital heart defects. During embryonic development, the heart initially forms as a straight tube. While it elongates, it acquires the shape of a rightward helix, a process referred to as heart looping. Previous work suggests that dynamic signaling in the heart precursor cells are essential for heart looping completion, but so far only a few genes have been identified as asymmetrically expressed. In order to investigate the dynamic spatiotemporal patterning of heart precursors, and screen for novel players in asymmetric heart morphogenesis, we aim to perform several transcriptomic analyses.

In recent years the concept that only adaptive immunity mediates memory responses has been challenged by several examples of innate immune memory, as shown for macrophages and NK cells. NK cells are innate lymphoid cells, with well-established roles in targeting cancer and virally infected cells, and in addition, are crucial players in antibacterial immunity. Similarly to what has been shown in response to virus, our work is revealing that NK cells maintain a memory of bacterial infection, as seen by a faster and more tailored response to subsequent infections. In response to viral infection, it has been shown that a certain subpopulation of NK cells become “memory” cells, which can expand and contract according to necessity. Similar studies in search of a memory population to bacterial infection have not been shown. Indeed, a major setback in such experiments is the lack of established markers for memory cells. Without a known receptor-ligand interaction, like what has been described for viruses, specific memory subpopulations are very difficult to identify by classical approaches. Single cell transcriptomics represents a novel approach that has been used to discover multiple immune subtypes and will provide a method to identify new sub-populations of memory cells within the NK cell pool.

Our project aims to study the consequences of the systemic inflammation on muscle stem cells (MuSCs). To address this question, we use two in vivo mouse models associated with high systemic inflammation, an acute viral respiratory infection, with the influenza virus, and a solid tumor growth. Both paradigms are associated with muscle catabolism, named cachexia, which is intensively studied in the cancer field. However, most studies are focusing on the muscle fibres, the contractile apparatus of the muscle. Our project is mainly focused on the study of the MuSCs, cells which give rise to skeletal muscle. We aim to characterize the molecular and functional modifications occurring in MuSCs when exposed to high inflammatory stress. RNAseq was performed for both paradigms to gain information on gene expression.

This projects aims at performing transcriptomics on mosquitoes infected with arboviruses such as Dengue or Zika virus.

Hematopietic stem cells (HSCs) are essential for supplying blood and immune cells of the vertebrate body. They are essentially formed during the embryonic time-period, from the wall of vessels and in particular the dorsal aorta. Their survival and self-renewal capabilities are depending on environmental conditions provided by hematopoietic niches among which the bone marrow microenvironment, in adult mammals. During embryonic development, HSC precursors home in transient niches; the main organ being the fetal liver. Currently, functional characteristics of developmental hematopoietic niches - including spatially-resolved biochemical and biomechanical specificities -, are lacking. This information is mandatory not only for fundamental aspects but also for the ultimate goal of producing in vitro HSCs with regeneration potential, for the purpose of regenerative medicine. The aim of our project is to characterize HSCs and niche cells at the single cell and spatial transcriptomic levels so as to identify spatially-restricted molecular components essential for HSC survival and stemness maintenance. To do so, we will use the zebrafish embryo that offers unique advantages in comparison to other vertebrates, in particular for investigating single cell HSC-niche interactions at the level of entire hematopoietic organs.

There are papain treated and papain+CPG treated group. From mouse lung tissue, I'd like to find out the target cell and their key molecule through scRNAseq and receptor-ligand analysis. When I get the target cell, I want to do ATACseq (or scATACseq) of target cell to see if memory of previous exposure to inflammatory stimuli represented by chromatin accessibility and which has something to do with 2nd exposure of same allergen.

Muscle stem cells in different anatomical locations are programmed with distinct upstream regulators prior to acquiring myogenic identity. This intriguing observation is most evident between cranial and trunk/limb muscles, and it correlates with the fact that cranial muscle stem cells are functionally more robust in terms of expansion capacity, resistance to stress, and engraftment efficiency, when compared to those in the limb. In this project we are using genomic approaches (sn-RNAseq and sc-ATACseq) and mouse models to study how the unique properties of cranial muscle groups are defined, and how they impact on myopathies where only subsets of skeletal muscles succumb to the disease process.

Kdm6b is a histone demethylase that specifically demethylates tri-methylated histone3 lysine27 (H3K27me3), a repressive mark responsible for gene silencing. Its removal by Kdm6b enables the transcription of specific target genes. Kdm6b is expressed in many cell types such as neurons, hematopoietic stem cells, leukocytes or fibroblasts. Type 2 Innate Lymphoid cells (ILC2) are the innate counterpart of Th2 lymphocytes enriched at the barrier surfaces where they play a role in tissue repair and anti-helminth defense. Previous work from our team showed that Kdm6b expression is upregulated in IL-33-activated ILC2s. Using a IL7RCre-Kdm6bflox mouse model that specifically deletes Kdm6b in all IL7R-expressing cells including ILC2, we observed decreased ILC2 numbers in bone marrow (BM) as well as in peripheral organs. qRT-PCR on sorted Kdm6b-deficient BM ILC2 precursors revealed decreased expression of several transcription factors as well as St2, the IL-33 receptor, all involved in ILC2 maturation and activation. Stimulation via intravenous (i.v) injections of IL-33 showed alteration of cytokine production in Kdm6b-deficient ILC2s compared to WT ILC2s, in BM as well as in the periphery. This observation was confirmed using an IL-33-dependent asthma model, the papain model, where Kdm6b-deficient lung ILC2s displayed decreased secretion of inflammatory cytokines. Altogether, our results suggest a role for Kdm6b in ILC2 maturation and Th2 pathway maintenance through the IL-33/ST2 axis.

The weaning reaction is a robust intestinal immune response that is induced by the expansion of the symbiotic microbiota during food diversification from milk to solid food. This weaning reaction is critical in setting the reactivity of the immune system in the long term to confer protection against inflammatory pathology in adulthood. With the aim to identify the key players in mediating this protective immunological imprinting, this project focuses on elucidating the role of myeloid cells and aims to decipher a potential protective signature at the transcriptomic and epigenetic level in the myeloid branch, notably the hematopoietic stem and myeloid progenitor cells, which are most likely to carry the long-term healthy imprinting.

It is increasingly clear that SARS-CoV-2 infection does not only affect the airways, but also the central nervous system (CNS) by interfering with neurons, glial cells, and the immune response in the brain, sometimes leading to long-lasting neurological signs, including anosmia and anxio-depressive symptoms. These and other persistent symptoms constitute a new entity that is currently called Long-COVID or Post-Acute COVID-19 Syndrome (PACS), however, it still lacks definitive diagnostic criteria and a universally accepted definition, as well a fully comprehensive view of its causative mechanisms. Our working hypothesis is that SARS-CoV-2 and/or the related inflammatory response can trigger a central mechanism in the CNS leading to the persistence of symptoms. The persistence of virus (or viral components, in particular viral RNA) along with the persistence of inflammation in the CNS may be an underlying cause for the occurrence of persistent symptoms in long COVID patients. Consequently, our main objective is to understand the origin and the mechanisms responsible for the persistent neurological symptoms such as long-term anosmia/ageusia or anxio-depressive symptoms post- SARS-CoV-2 infection, and we will focus on how viral neuroinvasion, neuroinflammation and/or signal transmitted by the neural route (lung-brain or visceral-brain axis) account for long-term post COVID-19 CNS alterations. At the end, the LongNeuroCOVID project should lead to several impactful results. From the scientific point of view, this work will improve the understanding of the general mechanisms involved in the spatio-temporal dynamics of SARS-CoV-2 infection, from entry in the CNS to causing long-term inflammation and CNS alterations. Describing these fundamental mechanisms should be of broader interest to the scientific community, as these results may be applicable to other, less understood, neuroinvasive pathogens. For public health, the characterization, in a relevant animal model and in an in vitro model of human neural networks, of the neurotropism of SARS-CoV-2 and its direct or indirect impact on long-term symptoms will be instrumental to the development of future therapies aimed at these clinical presentations. Finally, at the neuropsychiatric level, this study should provide new and interesting findings regarding the occurrence of anxio-depressive signs following the infection by SARS-CoV-2 and their relation with anosmia and neuroinflammation.

In the past few years, evidence that Natural Killer cells can acquire memory properties has challenged the dogma that only adaptive immunity mediates memory responses. This new property of NK cells is defined by a specific enhanced response upon re-exposure linked to substantial chromatin remodeling during NK clonal expansion, and to the acquisition of stable transcriptional changes. However, NK cell memory is poorly understood at the molecular level and exclusively described in response to viral infection. Whether NK cells could acquire memory during bacterial infections and what chromatin remodeling mechanisms are involved remained uninvestigated. However, in the lab, we have shown that following LPS-induced endotoxemia, NK cells acquire long-lasting memory properties associated with specific epigenetic modifications. In this project, using S pneumo infection as a model, we will perform single cell ATAC-seq coupled with transcriptomics (Multiomics) to identify the transcriptional program of the emerging subpopulations of memory NK cells and define its epigenomic landscape. With these data, we aim to identify chromatin rearrangements and gene expression patterns that are induced and maintained in the sub-population of memory NK cells. We believe that this project will enlighten our understanding of how NK cell memory identity and function is encoded at the chromatin level, a wide-open field of study.