Targeted cell elimination reveals an auxin-guided biphasic mode of lateral root initiation.

To sustain a lifelong ability to initiate organs, plants retain pools of undifferentiated cells with a preserved proliferation capacity. The root pericycle represents a unique tissue with conditional meristematic activity, and its tight control determines initiation of lateral organs. Here we show that the meristematic activity of the pericycle is constrained by the interaction with the adjacent endodermis. Release of these restraints by elimination of endodermal cells by single-cell ablation triggers the pericycle to re-enter the cell cycle. We found that endodermis removal substitutes for the phytohormone auxin-dependent initiation of the pericycle meristematic activity. However, auxin is indispensable to steer the cell division plane orientation of new organ-defining divisions. We propose a dual, spatiotemporally distinct role for auxin during lateral root initiation. In the endodermis, auxin releases constraints arising from cell-to-cell interactions that compromise the pericycle meristematic activity, whereas, in the pericycle, auxin defines the orientation of the cell division plane to initiate lateral roots.

Combined clonotyping and gene signatures of individual tumor-reactive CD8 T-cells define their functional relevance following peptide vaccination in melanoma patients

Novel cancer vaccines are capableto efficiently induce and boost humantumor antigen specific T-cells. However,the properties of these CD8T-cells are only partially characterized.For in depth investigation ofT-cells following Melan-A/MART-1peptide vaccination in melanoma patients,we conducted a detailed prospectivestudy at the single cell level.We first sorted individual human naiveand effector CD8 T-cells from peripheralblood by flow cytometry, andtested a modified RT-PCR protocolincluding a global amplification ofexpressed mRNAs to obtain sufficientcDNAfromsingle cells.We successfullydetected the expression ofseveral specific genes of interest evendown to 106-fold dilution (equivalentto 10-5 cell). We then analyzed tumor-specific effector memory (EM)CD8T-cell subpopulations ex vivo, assingle cells from vaccinated melanomapatients. To elucidate the hallmarksof effective immunity the genesignatures were defined by a panel ofgenes related to effector functions(e.g. IFN-, granzyme B, perforin),and individual clonotypes were identifiedaccording to the expression ofdistinct T-cell receptors (TCR). Usingthis novel single cell analysis approach,we observed that T-cell differentiationis clonotype dependent,with a progressive restriction in TCRBV clonotype diversity from EMCD28pos to EMCD28neg subsets. However,the effector function gene imprintingis clonotype-independent,but dependent on differentiation,since it correlates with the subset oforigin (EMCD28pos or EMCD28neg). We also conducted a detailedcomparative analysis after vaccinationwith natural vs. analog Melan-Apeptide. We found that the peptideused for vaccination determines thefunctional outcome of individualT-cell clonotypes, with native peptideinducing more potent effector functions.Yet, selective clonotypic expansionwith differentiation was preservedregardless of the peptide usedfor vaccination. In summary, the exvivo single cell RT-PCR approach ishighly sensitive and efficient, andrepresents a reliable and powerfultool to refine our current view of molecularprocesses taking place duringT-cell differentiation.

Profile of a serial killer: cellular and molecular approaches to study individual cytotoxic T-cells following therapeutic vaccination.

T-cell vaccination may prevent or treat cancer and infectious diseases, but further progress is required to increase clinical efficacy. Step-by-step improvements of T-cell vaccination in phase I/II clinical studies combined with very detailed analysis of T-cell responses at the single cell level are the strategy of choice for the identification of the most promising vaccine candidates for testing in subsequent large-scale phase III clinical trials. Major aims are to fully identify the most efficient T-cells in anticancer therapy, to characterize their TCRs, and to pinpoint the mechanisms of T-cell recruitment and function in well-defined clinical situations. Here we discuss novel strategies for the assessment of human T-cell responses, revealing in part unprecedented insight into T-cell biology and novel structural principles that govern TCR-pMHC recognition. Together, the described approaches advance our knowledge of T-cell mediated-protection from human diseases.

The behavioral relevance and top-down susceptibility of early, low level multisensory interactions in humans

Abstract (English)General backgroundMultisensory stimuli are easier to recognize, can improve learning and a processed faster compared to unisensory ones. As such, the ability an organism has to extract and synthesize relevant sensory inputs across multiple sensory modalities shapes his perception of and interaction with the environment. A major question in the scientific field is how the brain extracts and fuses relevant information to create a unified perceptual representation (but also how it segregates unrelated information). This fusion between the senses has been termed "multisensory integration", a notion that derives from seminal animal single-cell studies performed in the superior colliculus, a subcortical structure shown to create a multisensory output differing from the sum of its unisensory inputs. At the cortical level, integration of multisensory information is traditionally deferred to higher classical associative cortical regions within the frontal, temporal and parietal lobes, after extensive processing within the sensory-specific and segregated pathways. However, many anatomical, electrophysiological and neuroimaging findings now speak for multisensory convergence and interactions as a distributed process beginning much earlier than previously appreciated and within the initial stages of sensory processing.The work presented in this thesis is aimed at studying the neural basis and mechanisms of how the human brain combines sensory information between the senses of hearing and touch. Early latency non-linear auditory-somatosensory neural response interactions have been repeatedly observed in humans and non-human primates. Whether these early, low-level interactions are directly influencing behavioral outcomes remains an open question as they have been observed under diverse experimental circumstances such as anesthesia, passive stimulation, as well as speeded reaction time tasks. Under laboratory settings, it has been demonstrated that simple reaction times to auditory-somatosensory stimuli are facilitated over their unisensory counterparts both when delivered to the same spatial location or not, suggesting that audi- tory-somatosensory integration must occur in cerebral regions with large-scale spatial representations. However experiments that required the spatial processing of the stimuli have observed effects limited to spatially aligned conditions or varying depending on which body part was stimulated. Whether those divergences stem from task requirements and/or the need for spatial processing has not been firmly established.Hypotheses and experimental resultsIn a first study, we hypothesized that auditory-somatosensory early non-linear multisensory neural response interactions are relevant to behavior. Performing a median split according to reaction time of a subset of behavioral and electroencephalographic data, we found that the earliest non-linear multisensory interactions measured within the EEG signal (i.e. between 40-83ms post-stimulus onset) were specific to fast reaction times indicating a direct correlation of early neural response interactions and behavior.In a second study, we hypothesized that the relevance of spatial information for task performance has an impact on behavioral measures of auditory-somatosensory integration. Across two psychophysical experiments we show that facilitated detection occurs even when attending to spatial information, with no modulation according to spatial alignment of the stimuli. On the other hand, discrimination performance with probes, quantified using sensitivity (d'), is impaired following multisensory trials in general and significantly more so following misaligned multisensory trials.In a third study, we hypothesized that behavioral improvements might vary depending which body part is stimulated. Preliminary results suggest a possible dissociation between behavioral improvements andERPs. RTs to multisensory stimuli were modulated by space only in the case when somatosensory stimuli were delivered to the neck whereas multisensory ERPs were modulated by spatial alignment for both types of somatosensory stimuli.ConclusionThis thesis provides insight into the functional role played by early, low-level multisensory interac-tions. Combining psychophysics and electrical neuroimaging techniques we demonstrate the behavioral re-levance of early and low-level interactions in the normal human system. Moreover, we show that these early interactions are hermetic to top-down influences on spatial processing suggesting their occurrence within cerebral regions having access to large-scale spatial representations. We finally highlight specific interactions between auditory space and somatosensory stimulation on different body parts. Gaining an in-depth understanding of how multisensory integration normally operates is of central importance as it will ultimately permit us to consider how the impaired brain could benefit from rehabilitation with multisensory stimula-Abstract (French)Background théoriqueDes stimuli multisensoriels sont plus faciles à reconnaître, peuvent améliorer l'apprentissage et sont traités plus rapidement comparé à des stimuli unisensoriels. Ainsi, la capacité qu'un organisme possède à extraire et à synthétiser avec ses différentes modalités sensorielles des inputs sensoriels pertinents, façonne sa perception et son interaction avec l'environnement. Une question majeure dans le domaine scientifique est comment le cerveau parvient à extraire et à fusionner des stimuli pour créer une représentation percep- tuelle cohérente (mais aussi comment il isole les stimuli sans rapport). Cette fusion entre les sens est appelée "intégration multisensorielle", une notion qui provient de travaux effectués dans le colliculus supérieur chez l'animal, une structure sous-corticale possédant des neurones produisant une sortie multisensorielle différant de la somme des entrées unisensorielles. Traditionnellement, l'intégration d'informations multisen- sorielles au niveau cortical est considérée comme se produisant tardivement dans les aires associatives supérieures dans les lobes frontaux, temporaux et pariétaux, suite à un traitement extensif au sein de régions unisensorielles primaires. Cependant, plusieurs découvertes anatomiques, électrophysiologiques et de neuroimageries remettent en question ce postulat, suggérant l'existence d'une convergence et d'interactions multisensorielles précoces.Les travaux présentés dans cette thèse sont destinés à mieux comprendre les bases neuronales et les mécanismes impliqués dans la combinaison d'informations sensorielles entre les sens de l'audition et du toucher chez l'homme. Des interactions neuronales non-linéaires précoces audio-somatosensorielles ont été observées à maintes reprises chez l'homme et le singe dans des circonstances aussi variées que sous anes- thésie, avec stimulation passive, et lors de tâches nécessitant un comportement (une détection simple de stimuli, par exemple). Ainsi, le rôle fonctionnel joué par ces interactions à une étape du traitement de l'information si précoce demeure une question ouverte. Il a également été démontré que les temps de réaction en réponse à des stimuli audio-somatosensoriels sont facilités par rapport à leurs homologues unisensoriels indépendamment de leur position spatiale. Ce résultat suggère que l'intégration audio- somatosensorielle se produit dans des régions cérébrales possédant des représentations spatiales à large échelle. Cependant, des expériences qui ont exigé un traitement spatial des stimuli ont produits des effets limités à des conditions où les stimuli multisensoriels étaient, alignés dans l'espace ou encore comme pouvant varier selon la partie de corps stimulée. Il n'a pas été établi à ce jour si ces divergences pourraient être dues aux contraintes liées à la tâche et/ou à la nécessité d'un traitement de l'information spatiale.Hypothèse et résultats expérimentauxDans une première étude, nous avons émis l'hypothèse que les interactions audio- somatosensorielles précoces sont pertinentes pour le comportement. En effectuant un partage des temps de réaction par rapport à la médiane d'un sous-ensemble de données comportementales et électroencépha- lographiques, nous avons constaté que les interactions multisensorielles qui se produisent à des latences précoces (entre 40-83ms) sont spécifique aux temps de réaction rapides indiquant une corrélation directe entre ces interactions neuronales précoces et le comportement.Dans une deuxième étude, nous avons émis l'hypothèse que si l'information spatiale devient perti-nente pour la tâche, elle pourrait exercer une influence sur des mesures comportementales de l'intégration audio-somatosensorielles. Dans deux expériences psychophysiques, nous montrons que même si les participants prêtent attention à l'information spatiale, une facilitation de la détection se produit et ce toujours indépendamment de la configuration spatiale des stimuli. Cependant, la performance de discrimination, quantifiée à l'aide d'un index de sensibilité (d') est altérée suite aux essais multisensoriels en général et de manière plus significative pour les essais multisensoriels non-alignés dans l'espace.Dans une troisième étude, nous avons émis l'hypothèse que des améliorations comportementales pourraient différer selon la partie du corps qui est stimulée (la main vs. la nuque). Des résultats préliminaires suggèrent une dissociation possible entre une facilitation comportementale et les potentiels évoqués. Les temps de réactions étaient influencés par la configuration spatiale uniquement dans le cas ou les stimuli somatosensoriels étaient sur la nuque alors que les potentiels évoqués étaient modulés par l'alignement spatial pour les deux types de stimuli somatosensorielles.ConclusionCette thèse apporte des éléments nouveaux concernant le rôle fonctionnel joué par les interactions multisensorielles précoces de bas niveau. En combinant la psychophysique et la neuroimagerie électrique, nous démontrons la pertinence comportementale des ces interactions dans le système humain normal. Par ailleurs, nous montrons que ces interactions précoces sont hermétiques aux influences dites «top-down» sur le traitement spatial suggérant leur occurrence dans des régions cérébrales ayant accès à des représentations spatiales de grande échelle. Nous soulignons enfin des interactions spécifiques entre l'espace auditif et la stimulation somatosensorielle sur différentes parties du corps. Approfondir la connaissance concernant les bases neuronales et les mécanismes impliqués dans l'intégration multisensorielle dans le système normale est d'une importance centrale car elle permettra d'examiner et de mieux comprendre comment le cerveau déficient pourrait bénéficier d'une réhabilitation avec la stimulation multisensorielle.

The role of Pten in the Hematopoietic System and the Hematopoietic Stem Cells

Résumé Identification, localisation et activation des cellules souches hématopoiétiques dormantes in vivo Les cellules souches somatiques sont présentes dans la majorité des tissus régénératifs comme la peau, l'épithélium intestinal et le système hématopoiétique. A partir d'une seule cellule, elles ont les capacités de produire d'autres cellules souches du même type (auto-renouvellement) et d'engendrer un ensemble défini de cellules progénitrices différenciées qui vont maintenir ou réparer leur tissu hôte. Les cellules souches adultes les mieux caractérisées sont les cellules souches hématopoiétiques (HSC), localisées dans la moelle osseuse. Un des buts de mon travail de doctorat était de caractériser plus en profondeur la localisation des HSCs endogènes in vivo. Pour ce faire, la technique "label retaining assay", se basant sur la division peu fréquentes et sur la dormance des cellules souches, a été utilisée. Après un marquage des souris avec du BrdU (analogue à l'ADN) suivi d'une longue période sans BrdU, les cellules ayant incorporés le marquage ("label retaining cells" LCRs) ont pu être identifiées dans la moelle osseuse. Ces cellules LCRs étaient enrichies 300 fois en cellules de phenotype HSC et, en utilisant de la cytofluorométrie, il a pu être montré qu'environ 15% de toutes les HSCs d'une souris restent dormantes durant plusieures semaines. Ces HSCs dormantes à long terme ne sont probablement pas impliquées dans la maintenance de 'hématopoièse. Par contre, on assiste à l'activation rapide de ces HSCs dormantes lors d'une blessure, comme une ablation myéloide. Elles re-entrent alors en cycle cellulaire et sont essentielles pour une génération rapide des cellules progénitrices et matures qui vont remplacer les cellules perdues. De plus, la détection des LCRs, combinée avec l'utilisation du marqueur de HSCs c-kit, peut être utilisée pour la localisation des HSCs dormantes présentes dans la paroi endostéale de la cavité osseuse. De manière surprenante, les LCRs c-kit+ ont surtout étés trouvées isolées en cellule unique, suggérant que le micro-environement spécifique entourant et maintenant les HSCs, appelé niche, pourrait être très réduit et abriter une seule HSC par niche. Rôles complexes du gène supresseur de tumeur Pten dans le système hématopoiétique La phosphatase PTEN disparaît dans certains cancers héréditaires ou sporadiques humains, comme les gliomes, les cancers de l'utérus ou du sein. Pten inhibe la voie de signalisation de la PI3-kinase et joue un rôle clé dans l'apoptose, la croissance, la prolifération et la migration cellulaire. Notre but était d'étudier le rôle de Pten dans les HSC normale et durant la formation de leucémies. Pour ce faire, nous avons généré un modèle murin dans lequel le gène Pten peut être supprimé dans les cellules hématopoiétiques, incluant les HSCs. Ceci a été possible en croissant l'allèle conditionnelle ptenflox soit avec le transgène MxCre inductible par l'interféron α soit avec le transgène Scl-CreERt inductible par le tamoxifen. Ceci permet la conversion de l'allèle ptenflox en l'allèle nul PtenΔ dans les HSCs et les autres types cellulaires hématopoiétiques. Les souris mutantes Pten développent une splénomégalie massive causée par une expansion dramatiques de toutes les cellules myéloides. De manière interessante, alors que le nombre de HSCs dans la moelle osseuse diminue progressivement, le nombre des HSCs dans la rate augmente de manière proportionnelle. Etrangement, les analyses de cycle cellulaire ont montrés que Pten n'avait que peu ou pas d'effet sur la dormance des HSCs ou sur leur autorenouvellement. En revanche, une augmentation massive du niveau de la cytokine de mobilisation G-CSF a été détéctée dans le serum sanguin, suggérant que la suppression de Pten stimulerait la mobilisation et la migration des HSC de la moelle osseuse vers la rate. Finallement, la transplantation de moelle osseuse délétée en Pten dans des souris immuno-déficientes montre que Pten fonctionnerait comme un suppresseur de tumeur dans le système hématopoiétique car son absence entraîne la formation rapide de leucémies lymphocytaires. Summary Identification, localization and activation of dormant hematopoietic stun cells in vivo Somatic stem cells are present in most self-renewing tissues including the skin, the intestinal epithelium and the hematopoietic system. On a single cell basis they have the capacity to produce more stem cells of the same phenotype (self-renewal) and to give rise to a defined set of mature differentiated progeny, responsible for the maintenance or repair of the host tissue. The best characterized adult stem cell is the hematopoietic stem cell (HSC) located in the bone marrow. One goal of my thesis work was to further characterize the location of endogenous HSCs in vivo. To do this, a technique called "label retaining assay» was used which takes advantage of the fact that stem cells (including HSCs) divide very infrequently and can be dormant for months. After labeling mice with the DNA analogue BrdU followed by a long BrdU free "chase", BrdU "label retaining cells" (CRCs) could be identified in the bone marrow. These CRCs were 300-fold enriched for phenotypic HSCs and by using flow cytometry analysis it could be shown that about 15% of all HSCs in the mouse are dormant for many weeks. Our results suggest that these long-term dormant HSCs are unlikely to be involved in homeostatic maintenance. However they are rapidly activated and reenter the cell cycle in response to injury signals such as myeloid ablation. In addition, detection of LRCs in combination with the HSC marker c-Kit could be used to locate engrafted dormant HSCs close to the endosteal lining of the bone marrow cavities. Most surprisingly, c-Kit+LRCs were found predominantly as single cells suggesting that the specific stem cell maintaining microenvironment, called niche, has limited space and may house only single HSCs. Complex roles of the tumor suppressor gene Pten in the hematopoietic system. The phosphatase PTEN is lost in hereditary and sporadic forms of human cancers, including gliomas, endometrial and breast cancers. Pten inhibits the PI3-kina.se pathway and plays a key role in apoptosis, cell growth, proliferation and migration. Our aim was to study the role of Pten in normal HSCs and during leukemia formation. To do this, we generated a mouse model in which the Pten gene can be deleted in hematopoietic cells including HSCs. This was achieved by crossing the conditional ptenflox allele with either the interferona inducible MxCre or the tamoxifen inducible Scl-CreERT transgene. This allowed the conversion of the ptenflox allele into a pterr' null allele in HSCs and other hematopoietic cell types. As a result Pten mutant mice developed massive splenomegaly due to a dramatic expansion of all myeloid cells. Interestingly, while the number of bone marrow HSCs progressively decreased, the number of HSCs in the spleen increased to a similar extent. Unexpectedly, extensive cell cycle analysis showed that Pten had little or no effect on HSC dormancy or HSC self-renewal. Instead, dramatically increased levels of the mobilizing cytokine G-CSF were detected in the blood serum suggesting that loss-of Pten stimulates mobilization and migration of HSC from the BM to the spleen. Finally, transplantation of Pten deficient BM cells into immuno-compromised mice showed that Pten can function as a tumor suppressor in the hematopoietic system and that its absence leads to the rapid formation of T cell leukemia.

Rapid bacterial genome sequencing: methods and applications in clinical microbiology.

The recent advances in sequencing technologies have given all microbiology laboratories access to whole genome sequencing. Providing that tools for the automated analysis of sequence data and databases for associated meta-data are developed, whole genome sequencing will become a routine tool for large clinical microbiology laboratories. Indeed, the continuing reduction in sequencing costs and the shortening of the 'time to result' makes it an attractive strategy in both research and diagnostics. Here, we review how high-throughput sequencing is revolutionizing clinical microbiology and the promise that it still holds. We discuss major applications, which include: (i) identification of target DNA sequences and antigens to rapidly develop diagnostic tools; (ii) precise strain identification for epidemiological typing and pathogen monitoring during outbreaks; and (iii) investigation of strain properties, such as the presence of antibiotic resistance or virulence factors. In addition, recent developments in comparative metagenomics and single-cell sequencing offer the prospect of a better understanding of complex microbial communities at the global and individual levels, providing a new perspective for understanding host-pathogen interactions. Being a high-resolution tool, high-throughput sequencing will increasingly influence diagnostics, epidemiology, risk management, and patient care.

Detection of promoter activity by flow cytometric analysis of GFP reporter expression.

Low efficiency of transfection is often the limiting factor for acquiring conclusive data in reporter assays. It is especially difficult to efficiently transfect and characterize promoters in primary human cells. To overcome this problem we have developed a system in which reporter gene expression is quantified by flow cytometry. In this system, green fluorescent protein (GFP) reporter constructs are co-transfected with a reference plasmid that codes for the mouse cell surface antigen Thy-1.1 and serves to determine transfection efficiency. Comparison of mean GFP expression of the total transfected cell population with the activity of an analogous luciferase reporter showed that the sensitivity of the two reporter systems is similar. However, because GFP expression can be analyzed at the single-cell level and in the same cells the expression of the reference plasmid can be monitored by two-color fluorescence, the GFP reporter system is in fact more sensitive, particularly in cells which can only be transfected with a low efficiency.

Dormant and self-renewing hematopoietic stem cells and their niches.

In the mouse, over the last 20 years, a set of cell-surface markers and activities have been identified, enabling the isolation of bone marrow (BM) populations highly enriched in hematopoietic stem cells (HSCs). These HSCs have the ability to generate multiple lineages and are capable of long-term self-renewal activity such that they are able to reconstitute and maintain a functional hematopoietic system after transplantation into lethally irradiated recipients. Using single-cell reconstitution assays, various marker combinations can be used to achieve a functional HSC purity of almost 50%. Here we have used the differential expression of six of these markers (Sca1, c-Kit, CD135, CD48, CD150, and CD34) on lineage-depleted BM to refine cell hierarchies within the HSC population. At the top of the hierarchy, we propose a dormant HSC population (Lin(-)Sca1(+)c-Kit(+) CD48(-)CD150(+)CD34(-)) that gives rise to an active self-renewing CD34(+) HSC population. HSC dormancy, as well as the balance between self-renewal and differentiation activity, is at least, in part, controlled by the stem cell niches individual HSCs are attached to. Here we review the current knowledge about HSC niches and propose that dormant HSCs are located in niches at the endosteum, whereas activated HSCs are in close contact to sinusoids of the BM microvasculature.

Characterization of tumor reactive CD8 T cells induced by peptide vaccination in melanoma patients

ABSTRACT¦Naturally acquired tumor-specific T-cells can be detected in most advanced cancer patients.¦Yet, they often fail to control or eliminate the disease, in contrast to many virus-specific CD8¦T lymphocytes. Therapeutic vaccines aim at inducing and boosting specific T-cells mediated¦immunity to reduce tumor burden. The properties of CD8 T-cells required for protection from¦infectious disease and cancer are only partially characterized.¦The objectives of this study were to assess effector functions, stage of differentiation and¦clonotype selection of tumor-reactive T lymphocytes following peptide vaccination in¦melanoma patients over time. Results were compared to protective viral-specific T-cell¦responses found in healthy individuals. We also characterized dominant versus low/non¦dominant T-cell clonotypes with the aim to further understand the in vivo function of each set¦of frequency-based specific T-cells.¦Here we developed and applied a novel approach for molecular and functional analysis of¦single T lymphocytes ex vivo. T-cell receptor (TCR) clonotype mapping revealed rapid¦selection and expansion of co-dominant T-cell clonotypes, which made up the majority of the¦highly differentiated "effector" T-cells, but only 25% of the less differentiated "effectormemory"¦cells, mostly composed of non-dominant clonotypes. Moreover, we show that¦advanced effector cell differentiation was indeed clonotype-dependent. Surprisingly, however,¦the acquisition of effector functions (cytokine production, killing) was clonotype-independent.¦Vaccination of melanoma patients with native peptide induced competent effector function in¦both dominant and non-dominant clonotypes, suggesting that most if not all clonotypes¦participating in a T-cell response have the potential to develop equal functional competence.¦In contrast, many T-cells remained poorly functional after vaccination with analog peptide,¦despite similar clonotype-dependent differentiation. Our findings show that the type of¦peptide vaccine has a critical influence on the selection and functional activation of the¦clonotypic T-cell repertoire. They also show that systematic assessment of individual T-cells¦identifies the cellular basis of immune responses, contributing to the rational development of¦vaccines.

Simultaneous co-expression of memory- and effector-related genes by individual human CD8 T cells depends on antigen specificity and differentiation

Purpose/Objective: Phenotypic and functional T cell properties are usually analyzed at the level of defined cell populations. However, large differences between individual T cells may have important functional consequences. To answer this issue, we performed highly sensitive single-cell gene expression profiling, which allows the direct ex vivo characterization of individual virus- and tumor-specific T cells from healthy donors and melanoma patients. Materials and methods: HLA-A*0201-positive patients with stage III/ IV metastatic melanoma were included in a phase I clinical trial (LUD- 00-018). Patients received monthly low-dose of the Melan-AMART- 1 26_35 unmodified natural (EAAGIGILTV) or the analog A27L (ELAGIGILTV) peptides, mixed CPG and IFA. Individual effector memory CD28+ (EM28+) and EM28- tetramer-specific CD8pos T cells were sorted by flow cytometer. Following direct cell lysis and reverse transcription, the resulting cDNA was precipitated and globally amplified. Semi-quantitative PCR was used for gene expression and TCR BV repertoire analyses. Results: We have previously shown that vaccination with the natural Melan-A peptide induced T cells with superior effector functions as compared to the analog peptide optimized for enhanced HLA binding. Here we found that natural peptide vaccination induced EM28+ T cells with frequent co-expression of both memory/homing-associated genes (CD27, IL7R, EOMES, CXCR3 and CCR5) and effector-related genes (IFNG, KLRD1, PRF1 and GZMB), comparable to protective EBV- and CMV-specific T cells. In contrast, memory/homing- and effectorassociated genes were less frequently co-expressed after vaccination with the analog peptide. Conclusions: These findings reveal a previously unknown level of gene expression diversity among vaccine- and virus-specific T cells with the simultaneous co-expression of multiple memory/homing- and effector- related genes by the same cell. Such broad functional gene expression signatures within antigen-specific T cells may be critical for mounting efficient responses to pathogens or tumors. In summary, direct ex vivo high-resolution molecular characterization of individual T cells provides key insights into the processes shaping the functional properties of tumor- and virus-specific T cells.

Domain shuffling in a sensor protein contributed to the evolution of insect pathogenicity in plant-beneficial Pseudomonas protegens.

Pseudomonas protegens is a biocontrol rhizobacterium with a plant-beneficial and an insect pathogenic lifestyle, but it is not understood how the organism switches between the two states. Here, we focus on understanding the function and possible evolution of a molecular sensor that enables P. protegens to detect the insect environment and produce a potent insecticidal toxin specifically during insect infection but not on roots. By using quantitative single cell microscopy and mutant analysis, we provide evidence that the sensor histidine kinase FitF is a key regulator of insecticidal toxin production. Our experimental data and bioinformatic analyses indicate that FitF shares a sensing domain with DctB, a histidine kinase regulating carbon uptake in Proteobacteria. This suggested that FitF has acquired its specificity through domain shuffling from a common ancestor. We constructed a chimeric DctB-FitF protein and showed that it is indeed functional in regulating toxin expression in P. protegens. The shuffling event and subsequent adaptive modifications of the recruited sensor domain were critical for the microorganism to express its potent insect toxin in the observed host-specific manner. Inhibition of the FitF sensor during root colonization could explain the mechanism by which P. protegens differentiates between the plant and insect host. Our study establishes FitF of P. protegens as a prime model for molecular evolution of sensor proteins and bacterial pathogenicity.

Role of PPARβ in keratinocyte adhesion and migration during skin wound healing

RESUME L'homéostasie du tissu cutané est assurée par des interactions étroites entre les cellules le composant et par l'équilibre entre la différenciation et la prolifération des kératinocytes devant permettre un renouvellement constant du tissu. Après une blessure, les kératinocytes environnant la zone blessée sont activés par des cytokines. Ils acquièrent alors un phénotype migratoire qui s'accompagne d'une modulation de l'activité protéolytique de la matrice extra cellulaire, d'une modulation de la dynamique du cytosquelette d'active, de la polarisation de la cellule, de l'affaiblissement des contacts entre cellules et de changements dans leurs contacts avec la matrice extra cellulaire. PPARβ est un facteur de transcription activé par les acides gras et leurs dérivés. Il appartient à la famille des récepteurs nucléaires aux hormones et son expression est avérée dans les kératinocytes des follicules pileux et dans les kératinocytes inter-folliculaires activés par la blessure cutanée. Le rôle de PPARβ dans la peau est principalement lié à son effet protecteur contre l'apoptose ainsi qu'à son implication dans l'équilibre dynamique entre la prolifération et la différentiation des kératinocytes. L'objet de ce travail fut de déterminer le rôle de PPARβ dans les processus d'adhésion et de migration des kératinocytes activés durant la régénération de l'épithélium blessé. Nous avons montré que les souris dépourvues du gène codant pour PPARβ ont de sévères imperfections affectant la morphologie de l'épithélium. Ce phénotype est corrélé à la modulation imparfaite du réseau d'active chez les souris dépourvues de PPARβ, à un défaut de localisation de l'intégrine α3 impliquée dans les complexes induisant la migration cellulaire, ainsi qu'à la modulation de l'expression d'acteurs majeurs affectant l'activité protéolytique de la matrice extra cellulaire. En conclusion, nos résultats montrent que PPARβ est impliqué dans le contrôle de la dynamique du cytosquelette d'active et la polarisation des kératinocytes activés. PPARβ étant impliqué dans l'acquisition d'un phénotype migratoire, il est légitime de se demander s'il intervient de même dans d'autres types cellulaires, par exemple dans la transition épithéliale-mésenchymateuse durant le développement, ou encore la progression de cellules tumorales. SUMMARY Highly coordinated intercellular interactions and single cell metabolism ensure cell and tissue maintenance of the skin. Healing of a skin wound involves keratinocyte activation by cytokines and growth factors. Activated keratinocytes acquire a motile phenotype that requires extracellular matrix remodeling and subsequent ligand activation through proteolytic activity, as well as cytoskeletal reorganisation induced by the release of cell-cell junctions and by the signalling relayed via integrin receptors and their cytoplasmic adaptors. PPARβ is a transcription factor activated by polyunsaturated fatty acids and fatty acid derivatives which belong to the nuclear hormone receptor superfamily. It is expressed in activated keratinocytes where it plays an essential role in protecting them from apoptosis. In addition, it plays an important function in hair follicle morphogenesis at the time of elongation, via the regulation of the balance between keratinocyte differentiation and proliferation. The aim of the present work was to determine if PPARβ is also involved in the regulation of migration and adhesion properties of keratinocytes during skin wound healing. We have shown that wounded PPARβ null mice display severe abnormalities of the keratinocyte migratory layer as shown at the histological level and using three-dimensional reconstruction. This altered migratory phenotype is correlated to altered dynamic of the actin cytoskeleton network, impaired α3 integrin localisation in migrating keratinocytes and changes in the expression of a key actor involved in extracellular matrix proteolytic activity. These results show that PPARβ is implicated in the fine tuning of the actin network organisation and the polarisation of activated keratinocytes following an epithelial wound. Whether these mechanisms are also controlled by PPARβ in other cell types during epithelial mesenchymal transition or tumour cell progression is an interesting question to rise.

Combination of fluorescent reporters for simultaneous monitoring of root colonization and antifungal gene expression by a biocontrol pseudomonad on cereals with flow cytometry.

Some root-associated pseudomonads sustain plant growth by suppressing root diseases caused by pathogenic fungi. We investigated to which extent select cereal cultivars influence expression of relevant biocontrol traits (i.e., root colonization efficacy and antifungal activity) in Pseudomonas fluorescens CHA0. In this representative plant-beneficial bacterium, the antifungal metabolites 2,4-diacetylphloroglucinol (DAPG), pyrrolnitrin (PRN), pyoluteorin (PLT), and hydrogen cyanide (HCN) are required for biocontrol. To monitor host plant effects on the expression of biosynthetic genes for these compounds on roots, we developed fluorescent dual-color reporters suited for flow cytometric analysis using fluorescence-activated cell sorting (FACS). In the dual-label strains, the constitutively expressed red fluorescent protein mCherry served as a cell tag and marker for root colonization, whereas reporter fusions based on the green fluorescent protein allowed simultaneous recording of antifungal gene expression within the same cell. FACS analysis revealed that expression of DAPG and PRN biosynthetic genes was promoted in a cereal rhizosphere, whereas expression of PLT and HCN biosynthetic genes was markedly less sustained. When analyzing the response of the bacterial reporters on roots of a selection of wheat, spelt, and triticale cultivars, we were able to detect subtle species- and cultivar-dependent differences in colonization and DAPG and HCN gene expression levels. The expression of these biocontrol traits was particularly favored on roots of one spelt cultivar, suggesting that a careful choice of pseudomonad-cereal combinations might be beneficial to biocontrol. Our approach may be useful for selective single-cell level analysis of plant effects in other bacteria-root interactions.

MAR elements regulate the probability of epigenetic switching between active and inactive gene expression.

Gene expression often cycles between active and inactive states in eukaryotes, yielding variable or noisy gene expression in the short-term, while slow epigenetic changes may lead to silencing or variegated expression. Understanding how cells control these effects will be of paramount importance to construct biological systems with predictable behaviours. Here we find that a human matrix attachment region (MAR) genetic element controls the stability and heritability of gene expression in cell populations. Mathematical modeling indicated that the MAR controls the probability of long-term transitions between active and inactive expression, thus reducing silencing effects and increasing the reactivation of silent genes. Single-cell short-terms assays revealed persistent expression and reduced expression noise in MAR-driven genes, while stochastic burst of expression occurred without this genetic element. The MAR thus confers a more deterministic behavior to an otherwise stochastic process, providing a means towards more reliable expression of engineered genetic systems.

Cortical fast-spiking parvalbumin interneurons enwrapped in the perineuronal net express the metallopeptidases Adamts8, Adamts15 and Neprilysin.

The in situ hybridization Allen Mouse Brain Atlas was mined for proteases expressed in the somatosensory cerebral cortex. Among the 480 genes coding for protease/peptidases, only four were found enriched in cortical interneurons: Reln coding for reelin; Adamts8 and Adamts15 belonging to the class of metzincin proteases involved in reshaping the perineuronal net (PNN) and Mme encoding for Neprilysin, the enzyme degrading amyloid β-peptides. The pattern of expression of metalloproteases (MPs) was analyzed by single-cell reverse transcriptase multiplex PCR after patch clamp and was compared with the expression of 10 canonical interneurons markers and 12 additional genes from the Allen Atlas. Clustering of these genes by K-means algorithm displays five distinct clusters. Among these five clusters, two fast-spiking interneuron clusters expressing the calcium-binding protein Pvalb were identified, one co-expressing Pvalb with Sst (PV-Sst) and another co-expressing Pvalb with three metallopeptidases Adamts8, Adamts15 and Mme (PV-MP). By using Wisteria floribunda agglutinin, a specific marker for PNN, PV-MP interneurons were found surrounded by PNN, whereas the ones expressing Sst, PV-Sst, were not.