Volatile Constituents of Cucumis sativus: Differences Between Five Tropical Cultivars

International audienceCucumis sativus L. (cucumber), a creeping plant of the Cucurbitaceae family, is widely cultivated for its fruit. It is a tender annual with a rough, succulent, trailing stem and hairy leaves with three to five pointed lobes; the stem bears branched tendrils by which the plant can be trained on supports. Depending on the cultivar, the fruits are available in many different sizes, shapes, and colors. They range from thick, stubby little fruits (10–12 cm long) to Dutch greenhouse varieties (of up to 50 cm long)

Spleen-Resident CD4+ and CD4− CD8α− Dendritic Cell Subsets Differ in Their Ability to Prime Invariant Natural Killer T Lymphocytes

One important function of conventional dendritic cells (cDC) is their high capacity to capture, process and present Ag to T lymphocytes. Mouse splenic cDC subtypes, including CD8α+ and CD8α− cDC, are not identical in their Ag presenting and T cell priming functions. Surprisingly, few studies have reported functional differences between CD4− and CD4+ CD8α− cDC subsets. We show that, when loaded in vitro with OVA peptide or whole protein, and in steady-state conditions, splenic CD4− and CD4+ cDC are equivalent in their capacity to prime and direct CD4+ and CD8+ T cell differentiation. In contrast, in response to α-galactosylceramide (α-GalCer), CD4− and CD4+ cDC differentially activate invariant Natural Killer T (iNKT) cells, a population of lipid-reactive non-conventional T lymphocytes. Both cDC subsets equally take up α-GalCer in vitro and in vivo to stimulate the iNKT hybridoma DN32.D3, the activation of which depends solely on TCR triggering. On the other hand, and relative to their CD4+ counterparts, CD4− cDC more efficiently stimulate primary iNKT cells, a phenomenon likely due to differential production of co-factors (including IL-12) by cDC. Our data reveal a novel functional difference between splenic CD4+ and CD4− cDC subsets that may be important in immune responses

Role of splenic antigen-presenting cells in invariant Natural Killer T lymphocytes

La zone marginale de la rate apparaît comme un lieu stratégique de détection des antigènes et agents pathogènes véhiculés par le sang. Ces propriétés sont principalement liées à la présence de cellules appartenant au système immunitaire inné parmi lesquelles se trouvent des nombreuses cellules présentatrices d’antigènes (APC), comme les macrophages, les lymphocytes B de la zone marginale (MZB) ou encore les cellules dendritiques (DC). Ces cellules représentent une première ligne de défense contre les pathogènes véhiculés par le sang et sont importantes pour l’initiation des réponses immunes. Il a fortement était suggéré la localisation dans la zone marginale d’une autre population appartenant au système immunitaire inné : les lymphocytes T Natural Killer invariants ou iNKT. Ces lymphocytes T non conventionnels sont caractérisés par l’expression de marqueurs de cellules NK et de lymphocytes T conventionnels notamment le TCR. Contrairement aux lymphocytes T conventionnels, les iNKT reconnaissent des antigènes (Ag) lipidiques (d’origine exogène ou endogène) présentés par l’intermédiaire de la molécule CD1d exprimée à la surface des APC, notamment les DC. En réponse à ces lipides, et notamment l’α-galactosylceramide (α-GalCer), les cellules iNKT ont la capacité unique de sécréter rapidement de grandes quantités de cytokines immunomodulatrices comme l’IFN-γ et/ou l’IL-4 qui, en retour, permettent l’activation d’autres populations immunes telles que les DC, les cellules NK, les lymphocytes B et lymphocytes T conventionnels. Les DC, en tant qu’APC professionnelles, sont de puissantes cellules activatrices des lymphocytes T conventionnels mais également des iNKT. Cependant, bien que souvent souligné dans la littérature, le rôle des autres APC dans l’activation des lymphocytes T conventionnels mais surtout des iNKT restait relativement obscur lorsque ce travail de thèse a débuté. Parmi les APC, les MZB représentaient des cibles idéales puisqu’elles ont la particularité d’exprimer fortement les molécules de présentation telle que les molécules du CMH de classe II, la molécule CD1d mais aussi les molécules de co-stimulation. Nous avons donc débuté notre travail par l’étude du rôle des MZB dans l’activation des lymphocytes conventionnels et des iNKT. Nous montrons que les MZB sensibilisés avec un peptide de l’ovalbumine sont capables d’activer les lymphocytes T CD4+, dont la réponse est orientée vers un profil Th1 après l’activation des MZB par le CpG-ODN (agoniste du TLR-9). Ainsi, les MZB se comportent comme de véritables APC. Nous avons ensuite étudié l’activation des iNKT en réponse à lα’-GalCer. De façon surprenante, bien que les MZB expriment fortement la molécule CD1d, elles sont incapables d’activer in vitro les iNKT primaires en réponse l’α-GalCer libre. Elles sont cependant capables de présenter l’α-GalCer aux iNKT suggérant qu’il manque aux MZB des facteurs (solubles ou non) pour induire l’activation des iNKT. De façon intéressante, l’ajout de DC non sensibilisées restaure la production d’IFN-γ et d’IL-4 par les iNKT co-cultivés en présence de MZB sensibilisés avec l’α-GalCer. Nous montrons que les DC participent à cette activation via un mécanisme de présentation croisée mais également via l’apport de facteurs nécessaires aux MZB pour induire l’activation des iNKT. Il existe une réelle coopération entre ces deux types d’APC pour une activation optimale des iNKT. Finalement, nous montrons que les MZB sensibilisés avec l’α-GalCer induisent l’activation des lymphocytes iNKT et NK in vivo. Nous nous sommes ensuite concentrés sur les DC qui comme indiqué ci-dessus, sont des APC professionnelles. Cependant, dans la rate, les DC représentent une population très hétérogène dont le rôle de chaque sous-population notamment dans l’activation des iNKT était également très peu connu lorsque ce travail a débuté.The spleen, with its highly specialized lymphoid compartments, plays a central role in clearing blood-borne pathogens. Innate immune cells, that are mainly present in the marginal zone of the spleen, are strategically located to respond to blood-borne microorganisms and viruses. Among innate cells, macrophages and marginal zone B (MZB) cells represent the first line of defense against blood-borne pathogens and with dendritic cells (DC) are important for initiation of the immune response. Along with these populations of antigen-presenting cells (APC), it was also suggested that invariant Natural Killer T (iNKT), a population of innate-like T lymphocytes, were also located in the marginal zone of the spleen. Unlike conventional T lymphocytes, iNKT cells recognize exogenous and self (glyco)lipid antigens (Ag) presented by the non-classical class I Ag presenting molecule CD1d expressed on APC, in particular DC. Upon lipid recognition, in particular in response to the non-mammalian glycolipid, α-galactosylceramide (α-GalCer), iNKT cells have the unique capacity to rapidly produce large amounts of immunoregulatory cytokines, including IFN-γ and IL-4, which lead to downstream activation of other immune populations (DC, NK cells, B cells and conventional T cells). Through this property, iNKT cells influence the strength and quality of the ensuing immune response. Dendritic cells, as professional APC, are potent activators of conventional T lymphocytes and iNKT cells. When we started our PhD, the role of APC other than DC in the priming of T lymphocytes including iNKT cells remained unclear. Among them, MZB cells represented good candidates since they express high levels of MHC class II and CD1d molecules and their ability to activate and orientate conventional and innate-like T lymphocytes, such as iNKT cells, were elusive. We show that MZB cells, when loaded OVA peptide promote the release of IFN-γ and IL-4 by antigen specific CD4+ T lymphocytes and their stimulation with CpG-ODN biases them toward more Th1 inducers. Surprisingly, although able to activate iNKT hybridomas, MZB cells sensitized with free α-GalCer do not directly activate ex vivo sorted iNKT cells unless DC are added to the culture system. Dendritic cells help MZB cells to promote iNKT cell activation in part through α-GalCer cross-presentation and also through DC-expressed co-factors. Interestingly, MZB cells amplify the DC-mediated activation of iNKT cells and depletion of MZB cells from total splenocytes strongly reduces iNKT cell activation in response to α-GalCer. Thus, DC and MZB cells provide help to each other to optimize iNKT cell stimulation. Finally, in vivo transfer of α-GalCer-loaded MZB cells potently activates iNKT and NK cells. Thus, we show for the first time a role of MZB cell in iNKT cell activation in response to free α-GalCer, an important finding to better understand the modalities of iNKT cell activation. As mentioned above, DC are professional APC and thus are strong activators of conventional and unconventional T lymphocytes. However, DC in the spleen represent an heterogeneous cell population and when we started our study, the role of DC subsets in T lymphocyte priming was still unclear. Among DC subsets, we concentrated on the major splenic DC subset located in the marginal zone, the CD8α- DC. This DC subset was further subdivided in CD4+ and CD4- subtypes. We provide evidences that CD4+ and CD4- DC are equally efficient at priming CD4+ T lymphocytes when loaded with OVA peptide and whole OVA, leading to a mixed Th1/Th2 response, and also CD8+ T lymphocytes when pulsed with OVA peptide (but not whole OVA)

Rôle des cellules présentatrices d'antigènes spléniques dans l'activation des lymphocytes T Natural Killer invariants

The spleen, with its highly specialized lymphoid compartments, plays a central role in clearing blood-borne pathogens. Innate immune cells, that are mainly present in the marginal zone of the spleen, are strategically located to respond to blood-borne microorganisms and viruses. Among innate cells, macrophages and marginal zone B (MZB) cells represent the first line of defense against blood-borne pathogens and with dendritic cells (DC) are important for initiation of the immune response. Along with these populations of antigen-presenting cells (APC), it was also suggested that invariant Natural Killer T (iNKT), a population of innate-like T lymphocytes, were also located in the marginal zone of the spleen. Unlike conventional T lymphocytes, iNKT cells recognize exogenous and self (glyco)lipid antigens (Ag) presented by the non-classical class I Ag presenting molecule CD1d expressed on APC, in particular DC. Upon lipid recognition, in particular in response to the non-mammalian glycolipid, α-galactosylceramide (α-GalCer), iNKT cells have the unique capacity to rapidly produce large amounts of immunoregulatory cytokines, including IFN-γ and IL-4, which lead to downstream activation of other immune populations (DC, NK cells, B cells and conventional T cells). Through this property, iNKT cells influence the strength and quality of the ensuing immune response. Dendritic cells, as professional APC, are potent activators of conventional T lymphocytes and iNKT cells. When we started our PhD, the role of APC other than DC in the priming of T lymphocytes including iNKT cells remained unclear. Among them, MZB cells represented good candidates since they express high levels of MHC class II and CD1d molecules and their ability to activate and orientate conventional and innate-like T lymphocytes, such as iNKT cells, were elusive. We show that MZB cells, when loaded OVA peptide promote the release of IFN-γ and IL-4 by antigen specific CD4+ T lymphocytes and their stimulation with CpG-ODN biases them toward more Th1 inducers. Surprisingly, although able to activate iNKT hybridomas, MZB cells sensitized with free α-GalCer do not directly activate ex vivo sorted iNKT cells unless DC are added to the culture system. Dendritic cells help MZB cells to promote iNKT cell activation in part through α-GalCer cross-presentation and also through DC-expressed co-factors. Interestingly, MZB cells amplify the DC-mediated activation of iNKT cells and depletion of MZB cells from total splenocytes strongly reduces iNKT cell activation in response to α-GalCer. Thus, DC and MZB cells provide help to each other to optimize iNKT cell stimulation. Finally, in vivo transfer of α-GalCer-loaded MZB cells potently activates iNKT and NK cells. Thus, we show for the first time a role of MZB cell in iNKT cell activation in response to free α-GalCer, an important finding to better understand the modalities of iNKT cell activation. As mentioned above, DC are professional APC and thus are strong activators of conventional and unconventional T lymphocytes. However, DC in the spleen represent an heterogeneous cell population and when we started our study, the role of DC subsets in T lymphocyte priming was still unclear. Among DC subsets, we concentrated on the major splenic DC subset located in the marginal zone, the CD8α- DC. This DC subset was further subdivided in CD4+ and CD4- subtypes. We provide evidences that CD4+ and CD4- DC are equally efficient at priming CD4+ T lymphocytes when loaded with OVA peptide and whole OVA, leading to a mixed Th1/Th2 response, and also CD8+ T lymphocytes when pulsed with OVA peptide (but not whole OVA).La zone marginale de la rate apparaît comme un lieu stratégique de détection des antigènes et agents pathogènes véhiculés par le sang. Ces propriétés sont principalement liées à la présence de cellules appartenant au système immunitaire inné parmi lesquelles se trouvent des nombreuses cellules présentatrices d’antigènes (APC), comme les macrophages, les lymphocytes B de la zone marginale (MZB) ou encore les cellules dendritiques (DC). Ces cellules représentent une première ligne de défense contre les pathogènes véhiculés par le sang et sont importantes pour l’initiation des réponses immunes. Il a fortement était suggéré la localisation dans la zone marginale d’une autre population appartenant au système immunitaire inné : les lymphocytes T Natural Killer invariants ou iNKT. Ces lymphocytes T non conventionnels sont caractérisés par l’expression de marqueurs de cellules NK et de lymphocytes T conventionnels notamment le TCR. Contrairement aux lymphocytes T conventionnels, les iNKT reconnaissent des antigènes (Ag) lipidiques (d’origine exogène ou endogène) présentés par l’intermédiaire de la molécule CD1d exprimée à la surface des APC, notamment les DC. En réponse à ces lipides, et notamment l’α-galactosylceramide (α-GalCer), les cellules iNKT ont la capacité unique de sécréter rapidement de grandes quantités de cytokines immunomodulatrices comme l’IFN-γ et/ou l’IL-4 qui, en retour, permettent l’activation d’autres populations immunes telles que les DC, les cellules NK, les lymphocytes B et lymphocytes T conventionnels. Les DC, en tant qu’APC professionnelles, sont de puissantes cellules activatrices des lymphocytes T conventionnels mais également des iNKT. Cependant, bien que souvent souligné dans la littérature, le rôle des autres APC dans l’activation des lymphocytes T conventionnels mais surtout des iNKT restait relativement obscur lorsque ce travail de thèse a débuté. Parmi les APC, les MZB représentaient des cibles idéales puisqu’elles ont la particularité d’exprimer fortement les molécules de présentation telle que les molécules du CMH de classe II, la molécule CD1d mais aussi les molécules de co-stimulation. Nous avons donc débuté notre travail par l’étude du rôle des MZB dans l’activation des lymphocytes conventionnels et des iNKT. Nous montrons que les MZB sensibilisés avec un peptide de l’ovalbumine sont capables d’activer les lymphocytes T CD4+, dont la réponse est orientée vers un profil Th1 après l’activation des MZB par le CpG-ODN (agoniste du TLR-9). Ainsi, les MZB se comportent comme de véritables APC. Nous avons ensuite étudié l’activation des iNKT en réponse à lα’-GalCer. De façon surprenante, bien que les MZB expriment fortement la molécule CD1d, elles sont incapables d’activer in vitro les iNKT primaires en réponse l’α-GalCer libre. Elles sont cependant capables de présenter l’α-GalCer aux iNKT suggérant qu’il manque aux MZB des facteurs (solubles ou non) pour induire l’activation des iNKT. De façon intéressante, l’ajout de DC non sensibilisées restaure la production d’IFN-γ et d’IL-4 par les iNKT co-cultivés en présence de MZB sensibilisés avec l’α-GalCer. Nous montrons que les DC participent à cette activation via un mécanisme de présentation croisée mais également via l’apport de facteurs nécessaires aux MZB pour induire l’activation des iNKT. Il existe une réelle coopération entre ces deux types d’APC pour une activation optimale des iNKT. Finalement, nous montrons que les MZB sensibilisés avec l’α-GalCer induisent l’activation des lymphocytes iNKT et NK in vivo. Nous nous sommes ensuite concentrés sur les DC qui comme indiqué ci-dessus, sont des APC professionnelles. Cependant, dans la rate, les DC représentent une population très hétérogène dont le rôle de chaque sous-population notamment dans l’activation des iNKT était également très peu connu lorsque ce travail a débuté

Comparative genomic analysis of primary tumors and paired brain metastases in lung cancer patients by whole exome sequencing: a pilot study

International audienceLung cancer brain metastases (BMs) are frequent and associated with poor prognosis despite a better knowledge of lung cancer biology and the development of targeted therapies. The inconstant intracranial response to systemic treatments is partially due to tumor heterogeneity between the primary lung tumor (PLT) and BMs. There is therefore a need for a better understanding of lung cancer BMs biology to improve treatment strategies for these patients. We conducted a study of whole exome sequencing of paired BM and PLT samples. The number of somatic variants and chromosomal alterations was higher in BM samples. We identified recurrent mutations in BMs not found in PLT. Phylogenic trees and lollipop plots were designed to describe their functional impact. Among the 13 genes mutated in ≥ 1 BM, 7 were previously described to be associated with invasion process, including 3 with recurrent mutations in functional domains which may be future targets for therapy. We provide with some insights about the mechanisms leading to BMs. We found recurrent mutations in BM samples in 13 genes. Among these genes, 7 were previously described to be associated with cancer and 3 of them (CCDC178, RUNX1T1, MUC2) were described to be associated with the metastatic process

Response of Bractocera cucurbitae females (Diptera, Tephritidae) to cucurbit host fruit odors.

International audienceBactrocera cucurbitae (Diptera, Tephritidae) is one of the major pest for cucurbits (cucumber, zucchini, melon, etc.) and can cause nearly 90% production losses on Cucurbitaceae crops in La Réunion.The objective of the present study was to improve our knowledge of olfactory stimuli used by females of B. cucurbitae to locate cucurbit host plants. We firstly compared different laboratory devices (a wind tunnel, olfactometers with one or four arms and small test cages) for assessing fly responses to different host plants. In the wind tunnel, females showed no orientated response in the presence of host odours. By contrast, positive response in small test cages and olfactometers was recorded. This allowed us to measure the relative attractiveness of the odours of different wild and cultivated cucurbitaceae for B. cucurbitae females. In parallel, the volatile composition of the odours of fruit and flowers of these different cucurbits was characterised using headspace-solid phase microextraction (SPME)/gas chromatography-mass spectrometry (CG-MS). This study should allow us to assess the host preferences of the Melon fly and eventually identify some powerful kairomonal attractants for this pest

Targeted Delivery of α-Galactosylceramide to CD8α + Dendritic Cells Optimizes Type I NKT Cell–Based Antitumor Responses

International audienceImmunotherapy aiming at enhancing innate and acquired host immunity is a promising approach for cancer treatment. The invariant NKT (iNKT) cell ligand α-galactosylceramide (α-GalCer) holds great promise in cancer therapy, although several concerns limit its use in clinics, including the uncontrolled response it promotes when delivered in a nonvectorized form. Therefore, development of delivery systems to in vivo target immune cells might be a valuable option to optimize iNKT cell-based antitumor responses. Using dendritic cell (DC)-depleted mice, DC transfer experiments, and in vivo active cell targeting, we show that presentation of α-GalCer by DCs not only triggers optimal primary iNKT cell stimulation, but also maintains secondary iNKT cell activation after challenge. Furthermore, targeted delivery of α-GalCer to CD8α(+) DCs, by means of anti-DEC205 decorated nanoparticles, enhances iNKT cell-based transactivation of NK cells, DCs, and γδ T cells. We report that codelivery of α-GalCer and protein Ag to CD8α(+) DCs triggers optimal Ag-specific Ab and cytotoxic CD8(+) T cell responses. Finally, we show that targeting nanoparticles containing α-GalCer and Ag to CD8α(+) DCs promotes potent antitumor responses, both in prophylactic and in therapeutic settings. Our data may have important implications in tumor immunotherapy and vaccine development