Shape-Based Tracking Allows Functional Discrimination of Two Immune Cell Subsets Expressing the Same Fluorescent Tag in Mouse Lung Explant

Dendritic Cells (DC) represent a key lung immune cell population, which play a critical role in the antigen presenting process and initiation of the adaptive immune response. The study of DCs has largely benefited from the joint development of fluorescence microscopy and knock-in technology, leading to several mouse strains with constitutively labeled DC subsets. However, in the lung most transgenic mice do express fluorescent protein not only in DCs, but also in closely related cell lineages such as monocytes and macrophages. As an example, in the lungs of CX3CR1+/gfp mice the green fluorescent protein is expressed mostly by both CD11b conventional DCs and resident monocytes. Despite this non-specific staining, we show that a shape criterion can discriminate these two particular subsets. Implemented in a cell tracking code, this quantified criterion allows us to analyze the specific behavior of DCs under inflammatory conditions mediated by lipopolysaccharide on lung explants. Compared to monocytes, we show that DCs move slower and are more confined, while both populations do not have any chemotactism-associated movement. We could generalize from these results that DCs can be automatically discriminated from other round-shaped cells expressing the same fluorescent protein in various lung inflammation models

Étude numérique et expérimentale des déformations mécaniques induites par le soudage

L’objectif de la présente étude est de comparer les déformations induites par deux procédés de soudage : le laser impulsionnel et le laser fibre continu. L’étude s’applique au soudage de l’alliage TA6V, pour différentes géométries de plan de joint. Les travaux seront structurés en deux parties. Un volet expérimental est mené sur des assemblages de deux plaques, l’une étant bloquée et l’autre libre. Le soudage induit des déformations dans la pièce et celles-ci sont maximales si la pièce est libre de se déformer. Ces dernières sont mesurées in-situ (capteur mécanique). Un second volet vise à modéliser la génération des contraintes et déformations résiduelles par un calcul numérique. La nécessité de disposer de caractérisations expérimentales intervient à différents maillons de la chaîne de calcul. Tout d’abord, la phase d’interaction entre le faisceau et la matière étant extrêmement complexe à simuler, l’approche thermique se fonde sur un calcul de « flux thermique équivalent ». Ceci nécessite de disposer d’une micrographie caractéristique (vue en coupe de la zone fondue) qui sera utilisée pour identifier des termes de « sources thermiques » via une procédure d’optimisation. Une procédure spécifique sera développée pour chacun des procédés. Le calcul est mené dans le logiciel élément finis COMSOL Multiphysics. Les résultats du calcul (déplacement des points de la plaque libre) sont confrontés aux résultats expérimentaux tout au long de la simulation. Le modèle est applicable à des soudages mettant en jeu des géométries complexes, pour lesquelles les effets de bridage peuvent engendrer des contraintes résiduelles

Impact de la fissuration a chaud sur la tenue mécanique des liaisons heterogenes Ta/TA6V

Le soudage par laser impulsionnel est un procédé à haute densité d’énergie permettant de réaliser des assemblages hétérogènes mettant en jeu des matériaux aux propriétés thermo-physiques notablement différentes. Malheureusement ce type d’assemblage peut être fragilisé par des fissures dites à chaud, générées au cours du processus de solidification du fait des écarts importants entre les températures de fusion et entre les coefficients de dilatation des pièces à assembler. C’est typiquement le cas de l’assemblage hétérogène Ta-TA6V faisant l’objet de la présente étude. La première partie de l’étude est consacré à la problématique d’assemblage. Après avoir présenté les propriétés des matériaux à assembler, les mécanismes de fissuration à chaud sont détaillés. La seconde partie de cette étude est consacrée à l’impact des fissures sur la tenue mécanique des assemblages. En fatigue, la limite d’endurance de liaisons fissurées ou non à l’issue du procédé a tout d’abord été identifiée. L’impact des hétérogénéités chimiques sur le comportement mécanique sous chargement monotone a ensuite fait l’objet d’une attention particulière. Pour cela, des éprouvettes ont été instrumentées par lithographie (dépôt de plots permettant de mesurer la déformation) et les essais ont été réalisés in-situ dans un MEB afin de mettre en relation les sites d’activité plastique prépondérante avec la microstructure locale (orientation granulaire identifiée par EBSD et composition chimique). Une approche numérique au sein du logiciel COMSOL Multiphysics a permis de corréler les microstructures observées avec les cycles thermiques subis en différents point de la zone de soudage. Pour de forts incréments de déformations, une fissure s’initie dans le matériau le plus dur et le moins ductile : le TA6V. Cette fissure suit ensuite un chemin de propagation tortueux, directement lié à la présence d’interfaces entre des zones de type cellulaire dendritique et des amas riches en tantale

Emerging paradigms and questions on pro-angiogenic bone marrow-derived myelomonocytic cells.

Cancer-related inflammation has emerged in recent years as a major event contributing to tumor angiogenesis, tumor progression and metastasis formation. Bone marrow-derived and inflammatory cells promote tumor angiogenesis by providing endothelial progenitor cells that differentiate into mature endothelial cells, and by secreting pro-angiogenic factors and remodeling the extracellular matrix to stimulate angiogenesis though paracrine mechanisms. Several bone marrow-derived myelonomocytic cells, including monocytes and macrophages, have been identified and characterized by several laboratories in recent years. While the central role of these cells in promoting tumor angiogenesis, tumor progression and metastasis is nowadays well established, many questions remain open and new ones are emerging. These include the relationship between their phenotype and function, the mechanisms of pro-angiogenic programming, their contribution to resistance to anti-angiogenic treatments and to metastasis and their potential clinical use as biomarkers of angiogenesis and anti-angiogenic therapies. Here, we will review phenotypical and functional aspects of bone marrow-derived myelonomocytic cells and discuss some of the current outstanding questions

Reassignment of the murine 3′TRDD1 recombination signal sequence

International audienceT cell receptor genes are assembled in developing T lymphocytes from discrete V, D, and J genes by a site-specific somatic rearrangement mechanism. A flanking recombination signal, composed of a conserved heptamer and a semiconserved nonamer separated by 12 or 23 variable nucleotides, targets the activity of the rearrangement machinery to the adjoining V, D, and J genes. Following the rearrangement of V, D, or J genes, their respective recombination signals are ligated together. Although these signal joints are allegedly invariant, created by the head-to-head abuttal of the heptamers, some do exhibit junctional diversity. Recombination signals were initially identified by comparison and alignment of germ-line sequences with the sequence of rearranged genes. However, their overall low level of sequence conservation makes their characterization solely from sequence data difficult. Recently, computational analysis unraveled correlations between nucleotides at several positions scattered within the spacer and recombination activity, so that it is now possible to identify putative recombination signals and determine and predict their recombination efficiency. In this paper, we analyzed the variability introduced in signal joints generated after rearrangement of the TRDD1 and TRDD2 genes in murine thymocytes. The recurrent presence of identical nucleotides inserted in these signal joints led us to reconsider the location and sequence of the TRDD1 recombination signal. By combining molecular characterization and computational analysis, we show that the functional TRDD1 recombination signal is shifted inside the putative coding sequence of the TRDD1 gene and, consequently, that this gene is shorter than indicated in the databases

A novel population of human melanoma-specific CD8 T cells recognizes Melan-AMART-1 immunodominant nonapeptide but not the corresponding decapeptide.

HLA-A2-restricted cytolytic T cells specific for the immunodominant human tumor Ag Melan-A(MART-1) can kill most HLA-matched melanoma cells, through recognition of two naturally occurring antigenic variants, i.e., Melan-A nonamer AAGIGILTV and decamer EAAGIGILTV peptides. Several previous studies have suggested a high degree of TCR cross-reactivity to the two peptides. In this study, we describe for the first time that some T cell clones are exclusively nonamer specific, because they are not labeled by A2/decamer-tetramers and do not recognize the decamer when presented endogenously. Functional assays with peptides gave misleading results, possibly because decamers were cleaved by exopeptidases. Interestingly, nonapeptide-specific T cell clones were rarely Valpha2.1 positive (only 1 of 19 clones), in contrast to the known strong bias for Valpha2.1-positive TCRs found in decamer-specific clones (59 of 69 clones). Molecular modeling revealed that nonapeptide-specific TCRs formed unfavorable interactions with the decapeptide, whereas decapeptide-specific TCRs productively created a hydrogen bond between CDR1alpha and glutamic acid (E) of the decapeptide. Ex vivo analysis of T cells from melanoma metastases demonstrated that both nonamer and decamer-specific T cells were enriched to substantial frequencies in vivo, and representative clones showed efficient tumor cell recognition and killing. We conclude that the two peptides should be regarded as distinct epitopes when analyzing tumor immunity and developing immunotherapy against melanoma

In Vivo Persistence of Codominant Human CD8+ T Cell Clonotypes Is Not Limited by Replicative Senescence or Functional Alteration.

T cell responses to viral epitopes are often composed of a small number of codominant clonotypes. In this study, we show that tumor Ag-specific T cells can behave similarly. In a melanoma patient with a long lasting HLA-A2/NY-ESO-1-specific T cell response, reaching 10% of circulating CD8 T cells, we identified nine codominant clonotypes characterized by individual TCRs. These clonotypes made up almost the entire pool of highly differentiated effector cells, but only a fraction of the small pool of less differentiated "memory" cells, suggesting that the latter serve to maintain effector cells. The different clonotypes displayed full effector function and expressed TCRs with similar functional avidity. Nevertheless, some clonotypes increased, whereas others declined in numbers over the observation period of 6 years. One clonotype disappeared from circulating blood, but without preceding critical telomere shortening. In turn, clonotypes with increasing frequency had accelerated telomere shortening, correlating with strong in vivo proliferation. Interestingly, the final prevalence of the different T cell clonotypes in circulation was anticipated in a metastatic lymph node withdrawn 2 years earlier, suggesting in vivo clonotype selection driven by metastases. Together, these data provide novel insight in long term in vivo persistence of T cell clonotypes associated with continued cell turnover but not replicative senescence or functional alteration

Gene-specific signal joint modifications during V(D)J recombination of TCRAD locus genes in murine and human thymocytes

International audienceV(D)J recombination assembles functional T-cell receptor (TCR) genes from V, D and J components in developing thymocytes. Extensive processing of V, D and J extremities before they are ligated creates a high degree of junctional diversity which results in the generation of a large repertoire of different TCR chains. In contrast, the extremities of the intervening DNA segment, which bear the recombination signal sequences, are generally held to be monomorphic, so that signal joints (SJs) consist of the perfect head-to-head juxtaposition of recombination signal extremities. We analyzed the structure of SJs generated during the recombination of TCRAD locus genes in murine and human thymocytes. Junctional diversity resulting from N nucleotide additions or from N nucleotide additions and base loss was found for each type of SJ examined. Different patterns of processing/modification were found, suggesting that different enzymatic activities operate during recombination of TCRA and TCRD genes, although they are located within the same genetic locus. Recombination of the deltaRec-1 element generates a diverse repertoire of SJs exhibiting both combinatorial and junctional diversity in murine and human thymocytes. Therefore, SJ diversity appears to be an intrinsic feature of V(D)J recombination in unmanipulated thymocytes