Characterization of Stress Granules and P-Bodies during human T lymphocyte activation

Les Lymphocytes T constituent la principale ligne de défense contre les pathogènes persistant et les cellules tumorales. Ils possèdent à leur surface des récepteurs inhibiteurs (ou IR pour Inhibitory Receptors) qui servent originellement à assurer l'homéostasie de la réponse immunitaire et empêcher les réactions auto-immunes. Malheureusement, ces récepteurs se retrouvent exploités par les cellules tumorales qui tendent à surexprimer leurs ligands afin d'atténuer la réponse immunitaire antitumorale. Ces dernières années ont vu l'essor du "blocage des points de contrôle immunitaire" (ou ICB pour immune checkpoints blockade) utilisant des anticorps qui ciblent et inhibent les récepteurs inhibiteurs ou leurs ligands et empêchent l'échappement immunitaire. Bien que cette approche ait révolutionné le traitement de multiples cancers, la réponse objective des patients, tous cancers confondus, n'est que d'environ 30%. Pour améliorer l'efficacité de ce type de thérapie, il est nécessaire de mieux comprendre les mécanismes qui régulent l'expression des points de contrôle immunitaire dans les lymphocytes antitumoraux. Notre équipe a précédemment découvert que dans les lymphocytes T humains, les microtubules, les kinésines, et les granules de stress (SG) effectuent la régulation post-transcriptionnelle des ARN des IR. Au-delà de ces IR cependant, l'ensemble du transcriptome contrôlé par les SG reste inconnu, de même que les composants protéiques de ces complexes lymphocytaires. Il existe en réalité deux types majeurs de granules de ribonucléoprotéines : les SG susmentionnés et les processing-bodies (PB). Il s'agit essentiellement d'agrégats cytosoliques sans membrane de protéines et d'ARN. Pour caractériser leur constitution précise, j'ai développé un protocole de purification adapté aux cellules non adhérentes et à faible volume cytosolique que sont les lymphocytes T. Les protéomes et transcriptomes des SG et des PB purifiés des lymphocytes T ont ensuite été identifiés par spectrométrie de masse et séquençage d'ARN, respectivement. Ces analyses m'ont permis de démontrer que la constitution (protéique et ARN) des SG et des PB s'avère finalement très similaire dans les lymphocytes T naïfs ou activés, malgré leur organisation et leur répartition cytosolique différentes. De plus, cette thèse montre que les ARN enrichis dans ces granules de lymphocytes T codent essentiellement pour des protéines de réplication cellulaire, tandis que ceux appauvris contrôlent plutôt des fonctions traductionnelles et métaboliques. Ces résultats suggèrent un rôle clé de ces granules lors de l'activation lymphocytaire, en préservant la traduction immédiate des ARN nécessaires à l'activation cellulaire, mais en stockant ceux déterminant la phase proliférative ultérieure. De plus, les ARN des IR sont retrouvés dans les SG et les PB, indiquant que leur régulation est indépendante de -ou antérieure à- la formation de ces granules. Ainsi, la caractérisation complète de ces granules dans le contexte lymphocytaire T normal améliore notre compréhension de ce nouveau mécanisme régulateur, et permet désormais d'envisager son exploration comparative en pathologie humaine, notamment cancéreuse ou virale.T lymphocytes (TLs) are the main line of defense against persisting pathogens and tumor cells. They have Inhibitory Receptors (IRs) at their surface that ensure immune homeostasis and prevent the emergence of auto-immune reactivity. Unfortunately, these receptors are hijacked by cancer cells that tend to overexpress their ligands and are able to turn down the anti-tumor immune response by doing so. In the last decade, a new immuno-therapeutic strategy called "immune checkpoints blockade" (ICB) emerged, which consist in using antibodies that recognize either the IRs or their ligands, thereby preventing their interaction and subsequent. Although this approach revolutionized the treatment of multiple cancers, it only shows an overall objective response of ~30% in patients 1-year post-treatment. To improve this approach, it becomes necessary to better understand the mechanisms of regulation behind the expression of those IRs. An investigation led by our team demonstrated that the microtubule network, the kinesin 1 superfamily and the Stress Granules (SG) were key components of the post-transcriptional regulation of RNAs encoding for IRs in TLs. However, little is known about this mechanism, and the complete range of transcripts controlled by SGs is undefined, and so does it for the molecular components of these complexes in this context. There are in reality two types of ribonucleoprotein granules that can be found in T cells, known as the SGs and the Processing Bodies (PBs). Their assembly essentially rely on the accumulation of RNAs and proteins that seed the accretion of membraneless aggregates in the cytosol of TLs. To study their constitution, a protocol based on the literature was developed to properly suit the 'non-adherent' and 'low cytosolic volume' properties of T cells. Purified SGs and PBs contents were then assessed by transcriptomic and proteomic approaches including mass spectrometry and RNA sequencing, respectively. These analyses showed that despite a really different overall organization and distribution of SGs and PBs in TLs, their RNA and protein constitution was extremely similar in resting or 72h-activatedcells. Additionally, RNAs enriched in granules encode for proteins implicated in 'Cell Cycle', and 'DNA metabolism and Repair' while RNAs depleted from SGs and PBs encode for proteins implicated in 'metabolism' and 'translation'. This strongly suggest that SGs and PBs play an important role in the storage of RNA important for the incoming proliferative phase meanwhile RNAs encoding for cell adaptability are free and available for translation. Surprisingly, both SGs and PBs contain RNAs encoding for IRs. This suggest that the active mechanism regulating their expression involves SG proteins, but takes place before the formation or is independent of the granules themselves. The identification of the granules content allowed to put these complexes in the context of TLs, and open the path for a better understanding of their precise role in these cells. Pursuing the investigation of this regulatory mechanism behind the expression of IRs is still a priority and will allow to transpose this knowledge to pathological contexts like viral infection or cancer

Caractérisation des granules de stress et des P-bodies durant l'activation des lymphocytes T humains

T lymphocytes (TLs) are the main line of defense against persisting pathogens and tumor cells. They have Inhibitory Receptors (IRs) at their surface that ensure immune homeostasis and prevent the emergence of auto-immune reactivity. Unfortunately, these receptors are hijacked by cancer cells that tend to overexpress their ligands and are able to turn down the anti-tumor immune response by doing so. In the last decade, a new immuno-therapeutic strategy called "immune checkpoints blockade" (ICB) emerged, which consist in using antibodies that recognize either the IRs or their ligands, thereby preventing their interaction and subsequent. Although this approach revolutionized the treatment of multiple cancers, it only shows an overall objective response of ~30% in patients 1-year post-treatment. To improve this approach, it becomes necessary to better understand the mechanisms of regulation behind the expression of those IRs. An investigation led by our team demonstrated that the microtubule network, the kinesin 1 superfamily and the Stress Granules (SG) were key components of the post-transcriptional regulation of RNAs encoding for IRs in TLs. However, little is known about this mechanism, and the complete range of transcripts controlled by SGs is undefined, and so does it for the molecular components of these complexes in this context. There are in reality two types of ribonucleoprotein granules that can be found in T cells, known as the SGs and the Processing Bodies (PBs). Their assembly essentially rely on the accumulation of RNAs and proteins that seed the accretion of membraneless aggregates in the cytosol of TLs. To study their constitution, a protocol based on the literature was developed to properly suit the 'non-adherent' and 'low cytosolic volume' properties of T cells. Purified SGs and PBs contents were then assessed by transcriptomic and proteomic approaches including mass spectrometry and RNA sequencing, respectively. These analyses showed that despite a really different overall organization and distribution of SGs and PBs in TLs, their RNA and protein constitution was extremely similar in resting or 72h-activatedcells. Additionally, RNAs enriched in granules encode for proteins implicated in 'Cell Cycle', and 'DNA metabolism and Repair' while RNAs depleted from SGs and PBs encode for proteins implicated in 'metabolism' and 'translation'. This strongly suggest that SGs and PBs play an important role in the storage of RNA important for the incoming proliferative phase meanwhile RNAs encoding for cell adaptability are free and available for translation. Surprisingly, both SGs and PBs contain RNAs encoding for IRs. This suggest that the active mechanism regulating their expression involves SG proteins, but takes place before the formation or is independent of the granules themselves. The identification of the granules content allowed to put these complexes in the context of TLs, and open the path for a better understanding of their precise role in these cells. Pursuing the investigation of this regulatory mechanism behind the expression of IRs is still a priority and will allow to transpose this knowledge to pathological contexts like viral infection or cancer.Les Lymphocytes T constituent la principale ligne de défense contre les pathogènes persistant et les cellules tumorales. Ils possèdent à leur surface des récepteurs inhibiteurs (ou IR pour Inhibitory Receptors) qui servent originellement à assurer l'homéostasie de la réponse immunitaire et empêcher les réactions auto-immunes. Malheureusement, ces récepteurs se retrouvent exploités par les cellules tumorales qui tendent à surexprimer leurs ligands afin d'atténuer la réponse immunitaire antitumorale. Ces dernières années ont vu l'essor du "blocage des points de contrôle immunitaire" (ou ICB pour immune checkpoints blockade) utilisant des anticorps qui ciblent et inhibent les récepteurs inhibiteurs ou leurs ligands et empêchent l'échappement immunitaire. Bien que cette approche ait révolutionné le traitement de multiples cancers, la réponse objective des patients, tous cancers confondus, n'est que d'environ 30%. Pour améliorer l'efficacité de ce type de thérapie, il est nécessaire de mieux comprendre les mécanismes qui régulent l'expression des points de contrôle immunitaire dans les lymphocytes antitumoraux. Notre équipe a précédemment découvert que dans les lymphocytes T humains, les microtubules, les kinésines, et les granules de stress (SG) effectuent la régulation post-transcriptionnelle des ARN des IR. Au-delà de ces IR cependant, l'ensemble du transcriptome contrôlé par les SG reste inconnu, de même que les composants protéiques de ces complexes lymphocytaires. Il existe en réalité deux types majeurs de granules de ribonucléoprotéines : les SG susmentionnés et les processing-bodies (PB). Il s'agit essentiellement d'agrégats cytosoliques sans membrane de protéines et d'ARN. Pour caractériser leur constitution précise, j'ai développé un protocole de purification adapté aux cellules non adhérentes et à faible volume cytosolique que sont les lymphocytes T. Les protéomes et transcriptomes des SG et des PB purifiés des lymphocytes T ont ensuite été identifiés par spectrométrie de masse et séquençage d'ARN, respectivement. Ces analyses m'ont permis de démontrer que la constitution (protéique et ARN) des SG et des PB s'avère finalement très similaire dans les lymphocytes T naïfs ou activés, malgré leur organisation et leur répartition cytosolique différentes. De plus, cette thèse montre que les ARN enrichis dans ces granules de lymphocytes T codent essentiellement pour des protéines de réplication cellulaire, tandis que ceux appauvris contrôlent plutôt des fonctions traductionnelles et métaboliques. Ces résultats suggèrent un rôle clé de ces granules lors de l'activation lymphocytaire, en préservant la traduction immédiate des ARN nécessaires à l'activation cellulaire, mais en stockant ceux déterminant la phase proliférative ultérieure. De plus, les ARN des IR sont retrouvés dans les SG et les PB, indiquant que leur régulation est indépendante de -ou antérieure à- la formation de ces granules. Ainsi, la caractérisation complète de ces granules dans le contexte lymphocytaire T normal améliore notre compréhension de ce nouveau mécanisme régulateur, et permet désormais d'envisager son exploration comparative en pathologie humaine, notamment cancéreuse ou virale

Contrôle de l’activité lymphocytaire par les granules de stress

International audienc

Regulatory Mechanisms of Inhibitory Immune Checkpoint Receptors Expression

International audienc

Stress Granules in the Post-transcriptional Regulation of Immune Cells

International audienceImmune cell activation triggers transcriptional and translational programs eliciting cellular processes, such as differentiation or proliferation, essential for an efficient immune response. These dynamic processes require an intricate orchestration of regulatory mechanisms to control the precise spatiotemporal expression of proteins. Post-transcriptional regulation ensures the control of messenger RNA metabolism and appropriate translation. Among these post-transcriptional regulatory mechanisms, stress granules participate in the control of protein synthesis. Stress granules are ribonucleoprotein complexes that form upon stress, typically under control of the integrated stress response. Such structures assemble upon stimulation of immune cells where they control selective translational programs ensuring the establishment of accurate effector functions. In this review, we summarize the current knowledge about post-transcriptional regulation in immune cells and highlight the role of stress sensors and stress granules in such regulation

Provenance of early bronze age metal artefacts in western Switzerland using elemental and lead isotopic compositions and their possible relation with copper minerals of the nearby Valais

Ten Early Bronze Age (BzA1, 2200-2000 BC) copper artefacts from the central Valais region from Switzerland were studied for their elemental composition and lead isotope ratios. In order to answer the archaeological question of a local copper supply, a database for copper minerals across the Valais (Switzerland) has been established. This database contains 69 data on lead isotope ratios as well as additional information on the minerals and geochemical associations for copper minerals from 38 locations in the Valais. Comparisons of the artefacts were also made with data pertaining to minerals from various deposits from Europe and Anatolia taken from the literature. The provenance of the materials is very diverse. Some of the data are compatible with the data from the copper mineral deposits of the Valais region. Moreover, three copper lunulae were identified as possibly Tuscan, which demonstrates contacts between Italy and the Valais region. This pattern also establishes a multiplicity of provenances for the metal and cultural influences in the Alpine environment of the Rhone Valley of Switzerland at the beginning of the Early Bronze Age

Provenance of Early Bronze Age metal artefacts in Western Switzerland using elemental and lead isotopic compositions and their possible relation with copper minerals of the nearby Valais

Ten Early Bronze Age (BzA1, 2200e2000 BC) copper artefacts from the central Valais region from Switzerland were studied for their elemental composition and lead isotope ratios. In order to answer the archaeological question of a local copper supply, a database for copper minerals across the Valais (Switzerland) has been established. This database contains 69 data on lead isotope ratios as well as additional information on the minerals and geochemical associations for copper minerals from 38 locations in the Valais. Comparisons of the artefacts were also made with data pertaining to minerals from various deposits from Europe and Anatolia taken from the literature. The provenance of the materials is very diverse. Some of the data are compatible with the data from the copper mineral deposits of the Valais region. Moreover, three copper lunulae were identified as possibly Tuscan, which suggests contacts between Italy and the Valais region. This pattern also establishes a multiplicity of provenances for the metal and cultural influences in the Alpine environment of the Rhone Valley of Switzerland at the beginning of the Early Bronze Age.status: publishe