7- Henri Sandrine "Deciphering the Tumor-associated Myeloid Cell Heterogeneity: Toward a Better understanding and better manipulation of XCR1+ cDC1 and tumor-associated macrophages in vivo to improve cancer-immunotherapy and avoid auto-immunity"
Positionnement du projet :
Obesity is a worldwide epidemic associated with increased morbidity and mortality that imposes an enormous burden on individuals and public health. As a detrimental health condition, obesity is associated with multiple metabolic abnormalities, including insulin resistance, hyperglycemia, dyslipidemia, hypertension and non-alcoholic fatty liver disease, which are collectively defined as metabolic syndrome. Various recent studies indicate that obesity is a heritable condition that is highly sensitive to the environment. Thus, understanding the molecular roots of obesity is an important prerequisite to improve both prevention and management of this condition. Toll-like receptors (TLRs) are evolutionary conserved transmembrane receptors that drive innate and adaptive immunity through the recognition of various microbial components and endogenous molecules released by damaged or dead cells. Excessive nutrients can also be sensed by certain TLRs that lately have emerged as a critical link between inflammation and a contributor to obesity and insulin resistance. Recently, we showed that TLR8 restrains TLR7 function and that TLR8 deficiency in mice results in increased TLR7 signalling by dendritic cells (DCs) and development of lupus autoimmunity (Demaria et al, J Clin Invest, 2010; Desnues et al., PNAS, 2014). Interestingly, our preliminary in vivo data strongly indicate that TLR7 signalling and DCs can also contribute to obesity and metabolic syndrome.
Objectifs et Méthodes :
The main objective of project is to explore the novel idea that increased TLR7 signalling contributes to obesity and metabolic syndrome and emit the hypothesis that reducing TLR7 function may prevent the development of obesity and its harmful effects. We aim at exploring the contribution of TLR7 and DCs in the development of obesity, and metabolic syndrome using in vivo animal models and transcriptomics analysis and mobilizing a multidisciplinary consortium with expertise in immunology, metabolism and magnetic resonance imaging.
Résultats attendus :
The main expected outcome of the project is to provide the first proof of concept of the therapeutic potential of blocking TLR7 as a target to prevent inflammation in the metabolic disease of obesity. Our studies will advance our understanding regarding the contribution of TLR7 and dendritic cells in obesity and might lead to novel avenues for the prevention and treatment of this epidemic metabolic disorder that reduces life expectancy and has huge socioeconomic consequences.
Profil du candidat demandé :
We are looking for a highly motivated student with strong background in immunology and good level of English. Previous experience in mouse models will be a plus, but not mandatory.
Targeted modulation of translation is possibly the most ancient antimicrobial mechanism and is conserved across all kingdoms. In addition to the well-established global inhibition of translation provoked by infection, Rafael Argüello in the Pierre lab recently discovered a sequence-specific mechanism that affects mRNA translation. It appears that several posttranscriptional modifications (PTMs) in the anti-codon loop of tRNAs can be controlled by stress. This constitutes an additional layer of complexity to the genetic code, as it affects the relative efficiency with which certain synonymous codons within a given mRNA are translated. Using systems biology, genetics and molecular biology, this project aims to dissect this subtle mechanism of gene regulation. We will use two model systems, mammalian cells and the nematode C. elegans, to allow us to uncover the key conserved pathways involved.
We have already demonstrated an evolutionarily conserved pattern of codon usage in immune related genes in human, mouse and C. elegans that can be observed at the whole genome scale. Moreover, we observed that tRNAs from different mouse tissues contain strikingly different levels of PTMs and that genes involved in these PTMs are downregulated upon infection. We now wish to establish how the translation machinery changes to control the relative translation efficiency of synonymous codons upon infection. Therefore, using codon usage reporters and genetic tools (CRISPR-Cas9 in mammalian cells and forward and reverse genetics in C. elegans) combined with three original methods to monitor translational activity (1-3), we will identify the genes involved in regulating these tRNA PTMs. Their role in innate immunity will be addressed using a model of natural fungal infection established by the Ewbank lab (4-6). We hope to discover a novel gene expression regulatory mechanism that explains the enigmatic conservation of codon usage across genes with similar function. Although this project focuses on a novel type of gene expression regulation during immune stress, we anticipate that this mechanism could also be important for many other aspects of cell homeostasis.
1. Argüello et al. Journal of Cell Science (2018).
2. Argüello et al. “ZeNITH: a single cell based energetic metabolism profiling method to characterize samples ex-vivo” (INSERM transfert patent: EB17493).
3. Wulff et al., Biochemistry (2017)
4. Polanowska et al. Genetics (2018)
5. Lebrigand et al. PLoS Genetics (2016)
6. Zugasti et al. BMC Biology (2016)
A background in cell or molecular biology or biochemistry; experience with C. elegans or another model organism would be an advantage; good communication skills. The group is international, so proficiency in English is essential.
Oncogene activation in melanomas drives the expression of cytokines that trigger the recruitment, differentiation and functional reprogramming of immune cells, including immune-suppressive myeloid cells and tumour-associated macrophages, both being linked to suppression of T cell responses. NF-kB is the prototypical proinflammatory transcription factor and has been strongly linked with both intrinsic and extrinsic effects on tumourigenesis. However, its role on immune surveillance has not been elucidated yet.
Using an HRAS-driven inducible mouse melanoma model with genetically inactivated IKKb/NF-kB pathway, we showed that NF-kB-activation during melanomagenesis shapes the immune microenvironment in melanoma and governs immune-surveillance. IKKb -sufficient and -deficient melanomas show similar development in immune-deficient hosts. However, the growth of IKKb-deficient melanomas is efficiently controlled in immune-competent mice by tumour-specific CD8 T cells, while IKKb-sufficient tumours escaped immune-surveillance. Co-incidentally, recruitment of tumouricidal tumour-associated macrophages accumulate in IKKb-deficient melanomas, while those displaying a tumour-promoting program are gathered in IKKb-sufficient melanomas.
In this project, the main aims are:
1. Identification of NF-kB-regulated genes impacting immune surveillance
In-depth sequencing of IKKb -sufficient and -deficient melanomas are being conducted: the PhD candidate will be involved in the molecular and functional validation of some of the NF-kB-regulated and differentially expressed genes.
2. Role of the IKKb/NF-kB signalling in B-RAF-driven melanomas
Given the predominance of BRAF mutations in melanomas patients, mouse BRAF-driven melanoma cell lines with active or inactive IKKb/NF-kB will be established. The PhD candidate will conduct in vivo transplantation experiments to characterise both the immune responses raised against those distinct tumours and the tumour-associated immune-suppressive mechanisms: these analyses will use polychromatic flow cytometry including cell sorts, RNA extraction and high-throughput qPCR, multiplex assays for cytokines, confocal microscopy, in vitro functional assays.
PhD student candidates should have an appropriate academic record of high achievement with a strong desire and motivation to pursue a research career in immunology. The applicant should demonstrate the ability to work in collaboration, to contribute to a vibrant academic research environment, to communicate in English.
Nathalie AUPHAN-ANEZIN, email: email@example.com; tel: +33 (0)4 91 26 91 89
Toby LAWRENCE, email: firstname.lastname@example.org.
Centre d’Immunologie de Marseille-Luminy (CIML),
Parc Scientifique de Luminy, Case 906,
13288 Marseille, cedex 09, France.
Secondary lymphoid organs such as lymph nodes (LNs) are composed of leukocytes (~95%) and lymphoid stromal cells (~5%) that form the structural framework of these organs. Various specialized stromal cell subsets create dense three-dimensional (3D) cellular networks that control lymphocyte survival and migration, create the backbone of the LNs and provide the nutrients, soluble factors, and antigens to the various immune cells required for ‘immunological surveillance’ and the development of adaptive immune responses. Indeed, immune cells would not properly function or even survive without these stromal cell networks and a better understanding of their biology appears mandatory to our full comprehension of the immune system.
Three major subsets of LN macrophages (Mφ) have long been distinguished based on their differential locations and functions. Subcapsular sinus Mφ (SSM) reside in the floor of the subcapsular sinus, overlying follicles. They capture small amounts of lymph-borne substances and transfer them to the B cell follicles. SSM are also permissive to viral replication, a process that is thought to boost B and T cell activation. The Mφ associated with the medullary sinuses (MSM) are known for their ability to capture and destroy pathogens drained from sites of infection, a process that helps preventing systemic spread. Germinal-center Mφ, also called tingible-body Mφ, have a key role in the removal of apoptotic non-selected germinal center B cells through Mfge8-dependent phagocytosis. We have recently identified a new population of Mφ in the LN T-cell zone that we have named TZM (T Zone Mφ – Manuscript invited for revision by Immunity). TZM form a dense network of phagocytes in charge of clearing apoptotic cells, both at steady state and during an immune response, but the full spectrum of their biological functions remains to be characterized.
Generally, Mφ are endowed with well-known immunological functions. Equally important, but often overlooked, are the roles of Mφ in tissue development, homeostasis and repair through the regulation of non-hematopoietic stromal cells. These include functions in branching morphogenesis, neuronal patterning, angiogenesis, lymphangiogenesis, bone morphogenesis, adipogenesis, regeneration and fibrosis.
We hypothesize that LN Mφ regulate the development, function and remodeling of LN stromal cells and that in return, LN stromal cells control the immunobiological properties of LN Mφ.
1/ To delineate the regulatory roles of LN Mφ on LN stromal cells in mouse and human.
2/ To delineate the modulatory function of LN stromal cells on LN Mφ in mouse and human.
The candidate should hold a Master degree in Immunology. Knowledge in microscopy and mouse model is a plus but not mandatory. The candidate will integrate a young and dynamic team in which the PhD students are actively
supervised and trained by experienced researchers. Mouse work will be required. For more information : email@example.com
Positionnement du projet
Vaccination is widely considered one of the greatest medical achievements for preventing infectious diseases. The basis for most currently licensed human vaccines relies on the induction of high affinity antibodies by specific B cells that can neutralise pathogens in case of future exposures. However, the generation of long-lasting protective vaccines against certain respiratory pathogens, such as influenza virus and drug-resistant pneumococcus, has not been successful. Due to these limitations, respiratory infections remain the deadliest communicable disease and the highest cause of mortality in low-income countries.
The generation of protective antibodies upon pathogen encounter requires sequential rounds of diversification and selection of B cells in specialized structures known as germinal centers. Although the initial development of germinal centers is promptly induced in lymph nodes and spleen, these structures can also form directly at the sites of inflammation, like in the lung mucosa after respiratory infection. In particular, lung germinal centers represent an environment with large amounts of antigen and the unique ability to generate highly diverse antibodies.
The main objective of this PhD project is to unveil the early events of B cell activation upon pathogen exposure in the lung mucosa. We aim to decipher how air-borne pathogens are locally delivered to B cells in the lungs upon respiratory infection and whether microbial metabolites could act as a potential source of antigen to boost B cell responses. A precise delineation of the cellular and molecular mechanisms of B cell activation in response to respiratory infection will provide key medical insights towards the development of next-generation vaccines against air-borne pathogens.
The candidate will rely on complementary methodologies to investigate the early events of B cell responses taking place in the lung mucosa: from mouse genetics and in vivo viral or bacterial infectious models to single-cell RNA-seq, multicolor flow cytometry and confocal, light-sheet and 2-photon microscopy.
We expect to identify, at the cellular level, the processes involved in the delivery of pathogenic antigens to lung resident B cells upon viral and bacterial infection. This project aims to highlight that anatomy, and not just molecular components, matter in the efforts to develop new vaccination strategies against highly variable pathogens.
Profil du candidat demandé
We are looking for a highly motivated candidate to join our team. A background in immunology, good skills in flow cytometry and mouse work will be a plus. The candidate will benefit from the excellent scientific environment and cutting-edge technologies at the CIML.
- Hamon Yannick & He Hai-Tao "Unravel the link between TCR, IFN-γR and cholesterol/ABCs pathways in CD4 T cell"
Studies over the last decade have documented that cholesterol homeostasis not only drives innate immunity 1 but affects T cell biology. Augmenting cholesterol cell content can strongly heighten the T cell function in vivo and in vitro as well as promote the T cell anti-tumor responses 2-4 Reciprocally, TCR-stimulation can result in T cell cholesterol increase, partly due to lowering ABC transporter expression5. A feed forward mechanism is thus at play leading the specific, selective rapid and efficient T cell activation via its T-Cell Receptor (TCR). Furthermore, it has been shown that increasing membrane cholesterol diverts naïve CD4 T cell differentiation towards Th1 subset3. This occurred through potentiation of both TCR and cytokine receptor signaling, establishing an interesting positive feedback-signaling loop between cholesterol and Th1 polarization into CD4 T cell differentiation. IFN-γ has also been shown to be critical in the differentiation of naive CD4 T cells into Th1 CD4 T cells 6. Moreover, the IFN-γR activation of JAK/STAT signaling was found be linked to ABCA1 activity of cholesterol efflux7,8. Our lab has contributed to show that cholesterol dependent membrane nano-organization is critical for proper signal transduction through IFN-γR or TCR 9-11.
This proposal is to unravel the link between TCR, IFN-γR and cholesterol/ABCs pathways that would form an interconnected system controlling the CD4 T cell polarization program, from organism to molecule scale. This will be done through the analysis of a conditional KO mouse model leading to the deletion of cholesterol ABC transporters specifically in the T cell lineage (CD4+ as starting cell type). The project is additionally supported by CRISPR/Cas9 genome edited murine and human T-cell lines.
Analysis of the phenotype of these models will require at first sight flow cytometry, protein and lipid biochemistry, live cell imaging with conventional and advanced imaging tools such super-resolution microscopy (available in the host team and imaging facility).
The main expected result is that cholesterol imbalance due to ABCs K-O interfere with CD4+ T cell biology, anticipating a mild impact on thymic selection to a stronger one in peripheral T cells upon adaptive immune challenge. Our hypothesis is that the seminal defect relies at molecular level on membrane receptors in T cells, skewing immune synapse assembly and signal transduction leading to improper T cell response.
We are looking for a candidate with a strong background in molecular and cellular immunology, trained at least in flow cytometry and imaging, and motivated for interdisciplinary and advanced technological approaches.
1 Westerterp, M. et al. Cholesterol accumulation in dendritic cells links the inflammasome to acquired immunity. Cell metabolism 25, 1294-1304. e1296 (2017).
2 Armstrong, A. J., Gebre, A. K., Parks, J. S. & Hedrick, C. C. ATP-binding cassette transporter G1 negatively regulates thymocyte and peripheral lymphocyte proliferation. The journal of immunology 184, 173-183 (2010).
3 Surls, J. et al. Increased membrane cholesterol in lymphocytes diverts T-cells toward an inflammatory response. PLoS One 7, e38733, doi:10.1371/journal.pone.0038733 (2012).
4 Yang, W. et al. Potentiating the antitumour response of CD8+ T cells by modulating cholesterol metabolism. Nature 531, 651, doi:10.1038/nature17412 (2016).
5 Bensinger, S. J. et al. LXR signaling couples sterol metabolism to proliferation in the acquired immune response. Cell 134, 97-111, doi:10.1016/j.cell.2008.04.052 (2008).
6 Martín-Fontecha, A. et al. Induced recruitment of NK cells to lymph nodes provides IFN-γ for T H 1 priming. Nature immunology 5, 1260 (2004).
7 Hao, X.-r. et al. IFN-γ down-regulates ABCA1 expression by inhibiting LXRα in a JAK/STAT signaling pathway-dependent manner. Atherosclerosis 203, 417-428 (2009).
8 Pradel, L. C. et al. ATP-binding cassette transporter hallmarks tissue macrophages and modulates cytokine-triggered polarization programs. Eur J Immunol 39, 2270-2280, doi:10.1002/eji.200838867 (2009).
9 Blouin, C. M. et al. Glycosylation-dependent IFN-γR partitioning in lipid and actin nanodomains is critical for JAK activation. Cell 166, 920-934 (2016).
10 Chouaki-Benmansour, N. et al. Phosphoinositides regulate the TCR/CD3 complex membrane dynamics and activation. Sci Rep 8, 4966, doi:10.1038/s41598-018-23109-8 (2018).
11 Lasserre, R. et al. Raft nanodomains contribute to Akt/PKB plasma membrane recruitment and activation. Nat Chem Biol 4, 538-547, doi:10.1038/nchembio.103 (2008).
- Henri Sandrine "Deciphering the Tumor-associated Myeloid Cell Heterogeneity: Toward a Better understanding and better manipulation of XCR1+ cDC1 and tumor-associated macrophages in vivo to improve cancer-immunotherapy and avoid auto-immunity"
The focus of our research is to better understand the biology of the mononuclear phagocytic cells (dendritic cells and macrophages) in health and disease in order to manipulate their functional properties for disease treatment.
It is not yet completely understood how myeloid cell populations positively or negatively regulate the immune response needed to eliminate tumor cells and the lack of markers to map them has limited the study of their biological functions. A recent revolution in cancer therapy is the use of immune checkpoint blockade treatments which lead to disease regression and in some patients to long-term stabilization. Still, too many patients experience cancer progression and/or autoimmune syndromes. Hence other treatment options are required as well as a better understanding of how checkpoint blockade treatments regulate tumor-associated myeloid cells.
The aim of the PhD project is to pursue our strong investment and to capitalize on our strong expertise of mononuclear phagocytic cells in healthy tissues and on innovative myeloid-specific mouse models we generated to establish a detailed map of myeloid cells in melanomas and to understand their immunogenic or immunosuppressive functions in tumor models.
The PhD project will be divided in three axis :
- The first axis is aiming to perform in depth profiling of the myeloid cell subsets present in melanoma and therefore to identify them accurately and reliably based on selective expression of surface markers and to further decipher their origin (fate mapping) and unveil their biological functions (single cell RNA-sequencing).
- The second axis is aiming to better understand the role of XCR1+ cDC1 in tumor rejection in vivo as well as in the development of auto-immune syndromes in response to check-point blockade immunotherapy, using in vivo gene targeting with cre-lox based approach.
- The third axis part will assess the phenotypic and functional changes of the myeloid tumor atlas in response to check-point blockade immunotherapy.
The PhD results should provide predictive signatures for response to current and future anti-tumor treatments.
The project will combine various techniques such as cell biology, state-of-theart flow-cytometry, single-cell RNA sequencing and animal experimentation and will benefit from the supportive technical and scientific environment of CIML.
For this PhD project, we are looking for a highly motivated, curious and enthusiastic candidate with background in immunology and skills in computational analysis.
- Irla Magali "Characterizing a novel immune checkpoint controlling the suppressive activity of regulatory T cells"
Foxp3+ regulatory T cells (Tregs) constitute a subset of CD4+ T lymphocytes that are essential in controlling immune tolerance. This cell type possesses the extraordinary capacity to suppress hazardous autoreactive T cells that have escaped thymic selection and thereby prevents the development of inflammatory and autoimmune disorders. Tregs have potent anti-inflammatory and immunosuppressive properties through several mechanisms such as inhibitory cytokines, cytolysis of target cells and the modulation of antigen presentation.
Recent advances have proven the efficiency of Treg-cell based therapy to treat autoimmune disorders such as type 1 diabetes, rheumatoid arthritis and inflammatory bowel disease (IBD). Nevertheless, the quantity of Tregs required remains high, which is currently a major obstacle to the routine use of this therapy in the clinic. Therefore, the identification of molecules that boost Treg functions is currently under active investigations. Our laboratory identified a ligand that negatively regulates their suppressive activity. Remarkably, the adoptive transfer of Tregs deficient for this ligand shows a superior therapeutic benefit than wildtype Tregs to treat IBD and multi-organ autoimmunity.
The main objective of this PhD project is to bring the proof-of-concept that targeting this new natural immune checkpoint boosts Treg suppressive activity, which is a mandatory requisite before clinical investigations. We will evaluate the therapeutic potential of the genetically modified Tregs for this ligand using preclinical mouse models of autoimmunity. We will also determine by which mechanisms this novel immune checkpoint enhances mouse and human Treg activity already during their development in the thymus and later in periphery.
The candidate will silence the expression of this ligand by transducing ex vivo wildtype Tregs with lentiviral particles containing the corresponding short hairpin RNA (shRNA). The therapeutic benefit of these silenced Tregs will be assessed using preclinical mouse models of IBD and multi-organ autoimmunity. The molecular mechanisms and the action modes of these highly suppressive cells will be investigated by flow cytometry, culture assays, cell-sorting, RNA-sequencing, quantitative PCR and immunohistochemistry.
Results are expected to demonstrate that targeting this new natural immune checkpoint constitutes an efficient strategy to improve Treg cell-based therapy. We also expect to decipher the underlying molecular mechanism by which the identified ligand regulates Treg suppressive activity.
Profile of the PhD candidate
We are looking for a highly motivated candidate with a good background in Immunology. Good skills in flow cytometry and mouse models will be an advantage.
The candidate will join a dynamic team with extensive scientific exchange and will benefit from excellent scientific environment and state-of-art research facilities of the CIML.
- Lelouard Hugues "Rôle du système phagocytaire des plaques de Peyer dans l’initiation de la réponse immunitaire intestinale"
Positionnement du projet :
Les plaques de Peyer (PPs) sont les principales sentinelles du système immunitaire intestinal. Dispersées le long de l’intestin grêle, elles échantillonnent les antigènes, détectent les agents pathogènes et déclenchent une réponse immunitaire appropriée largement basée sur la génération de plasmocytes sécréteurs d’IgAs. Les PPs sont constituées de follicules de lymphocytes B séparées par des régions interfolliculaires riches en lymphocytes T. L’épithélium associé aux follicules contient des cellules épithéliales spécialisées dans l’échantillonnage, appelées cellules M, qui transportent les antigènes de la lumière intestinale vers le dôme sous-jacent. Ce dernier est enrichi en phagocytes spécialisés que nous avons récemment caractérisés par des approches de microscopie, cytométrie et RNASeq.
Objectifs : Notre but est de déterminer le rôle de ces phagocytes dans l’initiation de la réponse immunitaire mucosale sous différentes conditions de stimulation (adjuvants, infections, colonisation par le microbiote).
Méthodes : Nous étudions pour chaque population phagocytaire des PPs les capacités de capture d’antigène et d’activation des lymphocytes B et T. Nous étudions aussi dans des modèles infectieux ou de stimulation par adjuvants l’impact de différents modèles de déplétion des phagocytes sur la réponse immunitaire intestinale, en particulier le nombre et le phénotype des lymphocytes T, des lymphocytes B ainsi que la production d’IgAs spécifiques.
Résultats attendus : La caractérisation des fonctions de chaque population phagocytaire des PP devrait ouvrir la voie à leur manipulation afin de moduler la réponse immunitaire intestinale et proposer de nouvelles voies thérapeutiques.
Profil du candidat demandé : Master 2 immunologie ayant pratiqué l’expérimentation animale avec de bonnes connaissances en cytométrie.
- Memet Sylvie "Analysis of the signalling induced by Brucella oligosaccharides in the inflammatory response"
Positionnement du projet :
Immune defence against bacterial pathogens involves interactions between conserved microbial motifs and cell surface receptors (including TLRs, DC SIGN) as well as intracellular ones, such as the NOD proteins. In the infected host, the binding of microbial components to these receptors in immune cells triggers multiple effector functions that contribute to pathogen eradication. Brucella, a gram-negative bacterium, is the etiologic agent of brucellosis, a worldwide re-emerging zoonotic disease affecting farm mammals with serious economic loss. It is transmitted to humans via contaminated food or infected aerosols and leads to strong chronic inflammation. Brucella produces different types of oligosaccharides, such as LipoPolySaccharide (LPS) and cyclic-β-1,2glucan (CβG), that act as microbial motifs and induce an inflammation of variable intensity. Recent studies from the Gorvel group have shown that Brucella LPS is a poor TLR4 agonist compared to that of E. coli and plays a seminal role in the control of host immunity against Brucella infection. In contrast CβG, essential for Brucella intracellular survival, elicits potent immune stimulation without toxicity, and has thus been approved as a novel vaccine adjuvant. This PhD project will contribute to address both in vitro and in vivo the role of specific cellular receptors in the signalling triggered by these Brucella oligosaccharides.
Objectifs : This project aims to investigate the molecular and cellular mechanisms by which Brucella-derived oligosaccharides modulate the immune system and therefore unravel the regulatory pathways triggered and/or hijacked.
Méthodes : The candidate will learn a wide array of biochemical, cellular, molecular, histochemical and cytometry techniques, including mouse genetics, cell culture, FACS, immunofluorescence, immunoprecipitation, western, transcriptomic, RNA seq analyses. Several mouse mutants or transgenics, already established in our animal house facility, and in vitro derived dendritic cells or macrophages will be used.
Résultats attendus : The project will contribute to define both in vitro and in vivo the role and interplay of the transduction pathways triggered by Brucella oligosaccharides. A better knowledge of brucellosis, a devastating live stock disease and difficult to treat human zoonosis, may lead to new avenues of treatment and better prevention. Beyond brucellosis, unravelling CβG-induced signalling in immune cells will improve the preparation of this novel vaccine adjuvant.
Profil du candidat demandé : Motivated and enthusiastic candidate interested in studying host-pathogen interactions and more precisely keen on deciphering the regulatory networks triggered by Brucella oligosaccharides in vitro and in vivo in the mouse model.
Background. During thymopoiesis, TCRαβ fitness is tested by 2 processes: positive and negative selections. Thymocytes with no, or “unfit” receptors, i.e. displaying too low or too high affinity for self peptide-MHC, are eliminated through death by neglect and negative selection, respectively. While thymocytes with fit TCR (i.e. displaying intermediate affinity for p-MHC) are positively selected to further differentiate into mature thymocytes. Therefore, TCR signaling can induce two opposite outcomes during physiological thymocyte development: cell death or, cell survival/differentiation. Recently, we tackled the impact of TCR signalling in the development of T-cell acute lymphoblastic leukemias (T-ALL). T-ALL are malignant proliferation of T-cell progenitors abnormally arrested at various stages of thymopoiesis, they result from various combinations of genes alterations, among them, PTEN loss is one of the most potent and is frequently associated with TCRαβ+ T-ALL. We uncovered that TCRαβ signaling strength directly impacts on leukemia genesis. Indeed, our results indicate that Pten-deficient thymocytes harboring fit TCR are eliminated and thus do not develop leukemia while those with unfit TCR (that should have been eliminated during positive selection) are rescued from cell death and are selected for leukemogenesis.
Our main objectives are: to better define the cross-talk between Pten and TCR signaling networks and to decipher mechanisms determining Pten-deficient thymocytes fate toward cell survival/leukemia or cell death.
Methods. To achieve our objectives, we will apply a systems biology approach and thus, we will use multidisciplinary tools: mouse genetics, single-cell approaches, bioinformatics and mathematical modeling. To model Pten-mediated leukemogenesis, we use a Ptendel model (Pten is inactivated in thymocytes), which is crossed on different genetic backgrounds. Then, cells are analyzed at the single-cell (sc) level: scRNA-sequencing (scRNAseq), or CITE-Seq which enables to couple cellular phenotyping and transcriptomic. Sequencing data are analyzed using bioinformatic tools: reconstruction of transcriptional trajectory of thymocytes differentiation or undergoing leukemic transformation, gene regulatory network inference from single cell expression data and network dynamic analysis.
Expected results. This project will deliver a comprehensive set of single-cell expression data covering thymocytes development in a physiological, pre-tumoral and tumoral backgrounds. In fine, we expect to better understand the role of Pten during physiological and pathological development of T lymphocytes.
Candidate. We are looking for a molecular/cellular biologist interested in immunology and oncology. The student will be involved in mouse experiments, and besides classical molecular/cellular techniques he will develop & perform single-cell assays. The student will have the opportunity to interact with the bioinformatician of the team, in order to analyze his/her scRNAseq/CITE-Seq data.
Multicellular organisms are protected from the outside world by physical barriers, such as a cuticle in invertebrates, and a skin in vertebrates. They are essential during development, defining form, and are key to the maintenance of homeostasis. At all stages, they contribute to resistance against abiotic and biotic stresses. Any breach of their integrity allows potential microbes to enter. Animals therefore have mechanisms to deal with injury that involve both tissue repair and immune responses. The genetic model organism C. elegans is devoid of a sophisticated immune system or motile immune cells and relies on epidermal barrier epithelia to form a first line of defence against the environment. C. elegans therefore represents a perfect model to address the question of how epithelia directly sense and respond to damage.
C. elegans can be wounded either mechanically, genetically or through infection by the pathogenic fungus Drechmeria coniospora. Our previous functional studies, mainly through forward and reverse genetic screens, have allowed us to catalogue the genes involved in regulating the innate immune response in the C. elegans epidermis. We also characterized the cell biology of the wound response.
Our current priorities are understanding how innate immunity is triggered and how cell repair is linked to immune signalling. We recently found that the wound healing response repurposes developmental modules required during moulting. Further, we have found that the cytoskeletal changes induced by injury are intimately linked to the immune response. We will run a targeted genetic screen to find triggers of the response, hoping to identify chemo- or mechano-receptors in the epidermis. We will also explore a potential new mechanosensory structure in the skin, through cell biology, AFM and electron microscopy.
We believe that combining these approaches in this unique genetic model will lead to the identification of fundamental features in wound responses, and reinforce a commonality between tissue repair and development. In the long term, this knowledge may give insight into conserved damage-detection mechanisms fundamental to the maintenance of organismal homeostasis across species.
A background in cell or molecular biology, or genetics; experience with C. elegans or another model organism would be an advantage; good communication skills. The group is international, so proficiency in English is essential.
- Roulland Sandrine "Integrative study of B Cell Receptor signaling pathway in B cell lymphoma and therapeutic targeting"
Despite its indolent nature, Follicular Lymphoma - a Germinal Center (GC) B-cell derived blood cancer - remains a significant clinical burden as the majority of patients invariably relapse or transform into a drug resistant disease. Unfortunately, effective therapies for other B-cell lymphomas produce moderate clinical response in FL limiting their use in this setting. We currently lack an integrated understanding of the molecular mechanisms that sustain the abnormal proliferation/survival of FL cells and, consequently, no clear strategy for therapeutic intervention in FL.
Among breakthrough technologies that emerged in the past years, genome-wide CRISPR/Cas9 genetic screens revealed mechanisms of cell proliferation and survival, as well as drug resistance at unprecedented resolution. Signaling through the B-cell receptor (BCR) is central to the development of normal B-cells. In light of the numerous survival pathways activated downstream of the BCR, it has been shown that malignant B-cells could co-opt this receptor to promote their own growth. Our CRISPR screen survey in FL revealed a specific dependency towards a BCR activation mode that transduces ‘tonic’ signals and promotes cell survival via SYK/PI3K/AKT pathway. However, the modalities by which this ‘tonic’ signaling cascade exerts its action in the GC remain unexplored. In this thesis project, we thus aim to understand this form of BCR signaling in normal and pathological conditions in order to identify and optimize possible pharmacologic interventions to eradicate FL.
To that end, using state-of-the-art CRISPR-based genome-wide loss-of-function genomic screens, single-cell transcriptomics and proteomics, we wish to:
i) to comprehensively describe the genes that underlie resistance or promote response to existing BCR kinase therapies and identify in an unbiased manner novel genetic regulators of tonic B cell activation that when inhibited will negatively tune proliferation and enhance tumor killing
ii) to functionally study the transcriptional/signaling network that is regulated by those BCR interactors upon BCR engagement and the consequence of their genetic disruption either in vivo mouse B-cells and in vitro in human FL models.
We expect from this integrative approach to provide an unprecedented view of the BCR regulatory network in normal B and FL cells and a roadmap for the rational development of BCR inhibitor combinations in FL. In the long run, because we have a unique access to primary FL clinical samples (including clinical trials with SYK inhibitors), we plan to implement our basic discoveries into translational programs of clinical significance for FL.
To lead this project, we are looking for a candidate interested in onco-immunology, cellular biology and therapeutic innovations.
A l’origine de l’hématopoïèse se trouvent les cellules souches hématopoïétiques (CSH), des cellules rares et quiescentes. Depuis peu nous savons que lors d’une infection, les CSH sont capables de reconnaitre directement certains pathogènes ou cytokines inflammatoires produites. A la suite de cette stimulation, elles vont provisoirement se mettre à proliférer et se différencier vers le lignage myéloïde. Très récemment, nous avons montré qu’une infection, mimée par une injection de LPS, peut changer à long terme l’identité des CSH en modifiant leur code épigénétique, permettant ainsi à la CSH de répondre plus efficacement à une deuxième infection. Ce phénomène est appelé immunité entrainée et avait précédemment été mis en évidence dans les cellules immunitaires innées matures tel que les macrophages.
Le projet proposé vise à analyser si les modifications épigénétiques observées dans les CSH après une première stimulation infectieuse sont transmises aux macrophages matures et si ceci confère à ces macrophages une meilleure réponse fonctionnelle.
Objectif 1 : Analyse du paysage épigénétique, transcriptionnel et fonctionnel des macrophages dérivés in vitro de CSH pré-stimulées
Après avoir dérivés in vitro des macrophages à partir de CSH de souris ayant reçu ou non un stimulus infectieux (LPS, beta-glucan, polyI:C, cytokines, …), différentes analyses seront effectuées :
1/ analyse de l’épigénome par ATAC seq ainsi que des enhancers « poised » ou actifs par ChipSeq
2/ analyse en RNA-seq de la réponse transcriptionnelle globale en réponse à une nouvelle stimulation infectieuse (identique ou différente de la pré-stimulation)
3/ analyse de l’activité fonctionnelle (phagocytose, production de cytokines, activation …)
Objectif 2 : Analyse du paysage épigénétique, transcriptionnel et fonctionnel des macrophages dérivés in vivo de CSH pré-stimulées
Pour cela, des souris seront transplantées avec des CSH pré-stimulées ou non avec un stimuli infectieux, ce qui permet d’avoir in vivo au sein du même individu des macrophages matures provenant de CSH pré-stimulée ou non. Après re-stimulation avec un nouveau signal infectieux, des analyses similaires à celles de l’objectif 1 seront effectuées sur les macrophages ainsi dérivés in vivo: epigénome, transcriptome (après tri des cellules au FACS) et analyse de l’activité fonctionnelle qui pourra être réalisé à la fois in vitro et in vivo (phagocytose, présentation de l’antigène, production de cytokines, cicatrisation et remodelage tissulaire, …)
Objectif 3 : Validation fonctionnelle, in vivo, dans des conditions physiopathologiques réelles
Les macrophages alvéolaires (AM) sont les macrophages des poumons permettant d’éliminer le mucus présent à la surface des poumons. L’objectif ici est d’analyser la capacité des AM dérivant des CSH pré-stimulés à répondre à une infection. Pour cela, des souris transplantées avec des CSH entrainées ou naïves seront infectées avec le virus influenza PR8 (H1N1). La capacité des AM dérivant des CSH pré-stimulées à lutter contre le virus sera analysée.
Enfin, la perte des AM induite par l’infection grippale entraine également des dommages tissulaires importants dans les poumons. La régénération du tissu après infection sera analysée par histologie.
Profil du/de la candidat(e) :
Extrême motivation, rigueur, disponibilité et réactivité.
Excellente capacité à travailler en équipe, maitrise de l’anglais scientifique à l’écrit et à l’oral.
Une expérience en cytométrie multiparamétrique et en expérimentation animale serait un plus.
Les cellules dendritiques plasmacytoïdes (pDC) produisent plus de 95% des interférons de type I et III (IFN) au cours d’infections virales systémiques. Cependant, les individus déficients pour la détection des virus par les pDC ne sont pas plus susceptibles aux infections virales. Il n’est donc pas clair si, quand et comment les pDC promeuvent la défense antivirale. La production d’IFN est restreinte à une fraction minoritaire des pDC, dans les organes lymphoïdes, mais elle n’est pas observée dans les pDC du foie, des poumons ou des intestins. Dans ces organes les pDC seraient plutôt impliquées dans le maintien de l’homéostasie tissulaire et dans la tolérance aux allergènes alimentaires ou respiratoires. Une production exacerbée d’IFN par les pDC peut promouvoir des maladies autoimmunes. Les tumeurs pourraient détourner les fonctions tolérogènes des pDC afin d’échapper aux réponses immunitaires. Cependant, des études récentes ont remis en question l’identité même des pDC et, par conséquent, leur capacité à exercer certaines de ces fonctions. Afin de déterminer si, quand et comment les pDC exercent des effets bénéfiques ou délétères pour la santé, il faut pouvoir les manipuler spécifiquement chez la souris. Ce n’était auparavant pas possible par absence de marqueur spécifique. Nous avons fait sauter ce verrou technologique en utilisant une stratégie combinatoire à deux gènes pour spécifiquement détecter les pDC par fluorescence (pDC-TdT) ou les éliminer constitutivement (pDC-DTA).
Les trois objectifs poursuivis seront 1) cartographier la distribution tissulaire des pDC dans différentes conditions physiopathologiques; 2) comprendre comment l’environnement tissulaire et son état inflammatoire modèlent les fonctions des pDC; 3) étudier le rôle des pDC dans l’homéostasie tissulaire, et/ou la tolérance immunitaire et/ou l’immunosurveillance contre le cancer.
Objectif 1: microscopie confocale et cytométrie de flux multiparamétrique. Objectif 2: caractérisation transcriptionnelle et épigénétique au niveau de la cellule unique, suivie de tests fonctionnels selon les modules de gènes identifiés et leur lien avec des processus biologiques. Objectif 3: comparaison du développement de la maladie entre souris contrôles versus pDC-DTA, après infections virales primaires et secondaires, ou infections par des agents pathogènes intestinaux, ou greffe d’un cancer du sein, ou induction d’une inflammation chronique. Les techniques et modèles proposés sont déjà en place au laboratoire.
Nos études permettront de mieux comprendre les fonctions des pDC et leur régulation moléculaire. Ces connaissances permettront de développer de nouvelles stratégies thérapeutiques de ciblage des pDC pour traiter différentes pathologies.
Profil du candidat demandé
Très rigoureux, travailleur et passionné. Maitrise de la microscopie et/ou de la cytométrie de flux, et si possible, des techniques de base de biologie moléculaire.
Positionnement du projet: La peau constitue l’une des premières lignes de défense contre les menaces extérieures. Elle contient des cellules immunitaires « sentinelles » qui s’activent en cas de lésion. En particulier, les macrophages résidents du derme qui participent aux défenses contre les pathogènes et aux mécanismes de réparation tissulaire. Elle présente également un système nerveux sensoriel très développé, détectant des stimuli externes dont certains conduisent aux sensations de démangeaison et de douleur. Des études récentes ont mis en lumière un rôle clé des interactions neuro-immunes dans le contexte de réponses inflammatoires cutanées. En particulier, des neurones sensoriels exprimant le marqueur Nav1.8 peuvent moduler l'inflammation. Cependant les mécanismes moléculaires et cellulaires impliqués restent mal connus.
• Les neurones sensoriels NaV1.8+ qui innervent la peau comprennent deux grandes familles : des neurones peptidergiques, dont le rôle sur l'immunité est déjà relativement bien caractérisé, et des neurones non peptidergiques dont la fonction reste à explorer. Nos travaux précédents ont démontré un rôle régulateur d'une sous-population de neurones non-peptidergiques exprimant les marqueurs NaV1.8 et GINIP sur la réponse immune cutanée suite à une exposition de la peau aux rayons ultra-violets (UV)
Objectifs: Le projet de thèse sera de caractériser les mécanismes moléculaires et cellulaires impliqués dans le contrôle de la réponse immunitaire et inflammatoire par cette sous-population de neurones sensoriels (NaV1.8+ GINIP+).
Méthodes: La réponse immunitaire et les mécanismes de réparation tissulaire seront analysées suis à l'exposition de la peau à des agressions extérieures (plaies, exposition aux UV, infection) dans un modèle génétique de souris dépourvues de neurones NaV1.8+GINIP+.
Résultats attendus: Cette étude devrait permettre d'une part de mieux comprendre le rôle des interactions neuro-immunes dans les processus de réparation tissulaire et de réponse immunitaire cutanée et d'autre part d'ouvrir de nouvelles perspectives thérapeutiques.
Profil du candidat demandé: Le/la candidat-e devra avoir une formation théorique solide en immunologie et/ou neurosciences. Il/elle aura déjà acquis de l'expérience dans le domaine de l'expérimentation animale et devra déjà maitriser les techniques de cytométrie en flux, d'ELISA et de microscopie confocale.
Lymph nodes (LN) are highly organised structures to accomodate various interactions of the adaptive immune system. LN formation starts within the embryo by interaction of mesenchymal cells and hematopoietic cells named lymphoid tissue inducer (LTi) cells in specific niches. We have shown that vitamin A within the maternal diet affected LTi differentiation within the embryo, and this affected immunity of the offspring lifelong (vandePavert etal., Nature 2014). LTi cells were found to be part of the recently described and illustrous Innate Lymphoid Cell (ILC) family. The LTi cells are the first to appear during embryogenesis, well before the appearance of the other ILC family members. There is debate on whether these cells are true type 3 ILCs, or a more distant relative. Also, it not clear what the origin and ontogeny is of these cells. The fetal liver is an essential organ for hematopoiesis, and most publications use this organ as source for LTi cells. However, we observed that differentiation of fetal liver LTi cells is different than those obtained from the periphery, where the LN are formed. Therefore, in this project we set out to :
1) describe the exact ontogeny of the LTi cells in relation to the other ILCs and where these steps during differentiation take place.
2) to study the role of fetal liver stromal cells on the differentiation of precursor LTi cells.
We will use different mouse reporter and conditional knock-out models in order to lineage trace and detect the ILCs. The embryos and cells will be analysed using cytometry that we have setup already. Also, in collaboration with the bioinformatics platform at the CIML, we will analyse the cells using single cell sequencing. We will visualise the embryos using standard immunofluorescence with confocal microscopy and also image the whole embryo by lightsheet microscopy. We aim to provide a detailed ontogeny of the LTi and ILC family during embryogenesis.
We are looking for an enthusiastic student who is willing to make an extra effort in order to satisfy their curiosity. Preferably with a background in developmental biology or immunology. Experience with mice is an advantage.
Undergraduate and graduate internships
Every summer the CIML offers a limited number of unpaid internships to students from the first or second cycle level. To apply for these courses, you must send us a two paragraph letter outlining your interest in science (with emphasis on your experience and the immunology course(s) iin which you have participated) accompanied by a curriculum vitae.
Students should not contact the CIML but send their documents in PDF format to firstname.lastname@example.org between April 1st and May 1st, 2015. Applicants will be notified of the decision of the CIML by mail.