Abstract Systemic autoimmune connective tissue diseases (CTDs) are characterized by anti-nuclear antibodies, shared HLA-associated genetic risk, and frequent disease overlap, suggesting a central role for CD4+ T cells in pathogenesis. However, defining disease-driving CD4+ T-cell responses remains challenging due to their localization within lymphoid and affected tissues and the lack of approaches linking these responses to circulating counterparts. We combined [18F]-labeled thymidine PET/CT-guided tissue sampling, ex vivo antigen stimulation, and single-cell multiomics to characterize CD4+ T-cell responses in blood, PET-avid locoregional lymph nodes (LNs), and disease-affected tissues from patients with the immunologically distinct CTDs systemic sclerosis and Sjögren’s disease. PET-avid LNs from both diseases exhibited enhanced adaptive immune activity and contained an expanded population of interferon-stimulated gene (ISG)-expressing TRAIL+ CD4+ T cells. In Sjögren’s disease, active LNs and affected tissues harbored diverse effector CD4+ T-cell populations, including follicular and peripheral helper T cells and Th2/Th17 cells. In contrast, systemic sclerosis tissues lacked effector CD4+ T cells, while active LNs were enriched for naïve, regulatory, and TRAIL+ ISG CD4+ T cells. Antigen stimulation of peripheral blood mononuclear cells enriched for expanded effector CD4+ T-cell populations that shared activation profiles and clonal relationships with cells in LNs and affected tissues, many representing autoreactive antigen-specific T cells. TRAIL+ CD4+ T cells suppressed effector T-cell differentiation, autoreactive plasma cell generation, and autoantibody production in vitro, identifying a previously unrecognized immunoregulatory population. Together, this workflow enables comprehensive characterization of pathogenic and regulatory CD4+ T-cell responses across CTDs.

OBJECTIVES:Cytotoxic T cells and natural killer (NK) cells are central effector cells in cancer and infections. Their effector response is regulated by activating and inhibitory receptors. The regulation of these cells in systemic autoimmune diseases such as systemic sclerosis (SSc) is less defined. METHODS:We conducted ex vivo analysis of affected skin and blood samples from 4 SSc patient cohorts (a total of 165 SSc vs 80 healthy individuals) using single-cell transcriptomics, flow cytometry and multiplex immunofluorescence staining. We further analysed the effects of costimulatory modulation in functional assays, and in a severely affected SSc patient who was treated on compassionate use with a novel anti-CD3/CD7 immunotoxin treatment. RESULTS:Here, we show that SSc-affected skin contains elevated numbers of proliferating T cells, cytotoxic T cells and NK cells. These cells selectively express the costimulatory molecule CD7 in association with cytotoxic, proinflammatory and profibrotic genes, especially in recent-onset and severe disease. We demonstrate that CD7 regulates the cytolytic activity of T cells and NK cells and that selective depletion of CD7+ cells prevents cytotoxic cell-induced fibroblast contraction and inhibits their profibrotic phenotype. Finally, anti-CD3/CD7 directed depletive treatment eliminated CD7+ skin cells and stabilised disease manifestations in a severely affected SSc patient. CONCLUSION:Together, the findings imply costimulatory molecules as key regulators of cytotoxicity-driven pathology in systemic autoimmune disease, yielding CD7 as a novel target for selective depletion of pathogenic cells.

The aerial surface of the plant (phyllosphere) is the habitat of complex microbial communities. These communities may have profound effects on host plant health and its performance traits. When breeding new cultivars, i.e. the aerial component of a grape plant, one can simply ignore the phyllosphere in breeding schemes if its composition is mainly dependent on the environment. It is considered an important component if the genotype is the main driver of the phyllosphere composition. In order to answer this question, we have analysed several factors influencing the structure of the phyllosphere microbial community. Using amplicon sequencing of the 16S rRNA gene and of the internal transcribed spacer (ITS), we explored the microbial diversity at genus level for both bacteria and fungi present in the phyllosphere of leaves and grape berries. We analysed it on different grape taxonomic level (between five Vitis species or a set of Vitis vinifera cultivars chosen to represent the three genetic pools of the species), for different years and on five commercially important varieties of Vitis vinifera that were sampled from three different French terroirs. Our results indicated the presence of complex microbial diversity and assemblages in the phyllosphere. A significant effect of several factors (organ, grape species, growing year and terroir) on taxa abundance was observed with varying degrees of effect. At a given location, genotypes have an impact on microbial assemblage in the phyllosphere of leaf and berries, most pronounced on fruits but the effect of terroir was much stronger than the cultivar identity when the leaf phyllosphere of five grapevine varieties grown in different agro-climatic zones was compared. Limitations of the study as well as implied consequences of this work will be discussed.

Hematopoiesis plays a critical role in maintaining a diverse pool of blood cells throughout human life. Despite recent efforts with single-cell data analyses, the nature of the early cell fate decisions and compartmentalization of progenitors remains contentious due to the sparsity and noise of the data. Using publically available single-cell RNA-Seq hematopoietic data from bone marrow, with three different matrix factorization approaches to recover associated gene modules from cell clusters reveals a tri-directional and hierarchically-structured transcriptional landscape of hematopoietic differentiation. We also devised a bootstrap method, which in combination with the above can better characterize the progenitor compartments and retrieve cellular hierarchies.

Background Acute myeloid leukemia (AML) is a heterogeneous and aggressive blood cancer that results from diverse genetic aberrations in the hematopoietic stem or progenitor cells (HSPCs) leading to the expansion of blasts in the hematopoietic system. The heterogeneity and evolution of cancer blasts can render therapeutic interventions ineffective in a yet poorly understood patient-specific manner. In this study, we investigated the clonal heterogeneity of diagnosis (Dx) and relapse (Re) pairs at genetic and transcriptional levels, and unveiled the underlying pathways and genes contributing to recurrence. Methods Whole-exome sequencing was used to detect somatic mutations and large copy number variations (CNVs). Single cell RNA-seq was performed to investigate the clonal heterogeneity between Dx-Re pairs and amongst patients. Results scRNA-seq analysis revealed extensive expression differences between patients and Dx-Re pairs, even for those with the same -presumed- initiating events. Transcriptional differences between and within patients are associated with clonal composition and evolution, with the most striking differences in patients that gained large-scale copy number variations at relapse. These differences appear to have significant molecular implications, exemplified by a DNMT3A/FLT3-ITD patient where the leukemia switched from an AP-1 regulated clone at Dx to a mTOR signaling driven clone at Re. The two distinct AML1-ETO pairs share genes related to hematopoietic stem cell maintenance and cell migration suggesting that the Re leukemic stem cell-like (LSC-like) cells evolved from the Dx cells. Conclusions In summary, the single cell RNA data underpinned the tumor heterogeneity not only amongst patient blasts with similar initiating mutations but also between each Dx-Re pair. Our results suggest alternatively and currently unappreciated and unexplored mechanisms leading to therapeutic resistance and AML recurrence.

One of the biggest demanding situations for food security in the 21st century is to enhance crop yield stability through the improvement of diseases-resistant crops. Managing plant health is a major challenge for modern food production and compounded by the lack of common ground among the many disease control disciplines involved. All plants simultaneously engage with billions of microbes which can be collectively referred to as the plant microbiome. Most microbes inside the plant microbiome are harmless or even beneficial to the plant as they promote plant growth or provide protection in opposition to diseases. However, some of these microbes also cause disease with devastating effects on crop yields. To prevent pathogen infection, plants have evolved an advanced innate immune system that recognizes conserved cell surface molecules that most pathogen possesses. Activation of the plant immune system stops the invading pathogen, however this comes with fitness cost that significantly reduces plant growth and leads to yield penalty. Apart from their innate immune system controlling pre-programmed defense reactions, plants can also increase the responsiveness of their immune system in response to selected environmental signals. This phenomenon is known as “defense priming”. Although defense priming rarely provides full protection, its broad-spectrum effectiveness, low-fitness cost, long‐lasting durability and inherited to future generations make it attractive for sustainable crop protection.

Impacts of plant genotype on microbial assemblage in the phyllosphere (above-ground parts of plants, which predominantly consists of the set of photosynthetic leaves) of Vitis vinifera cultivars have been studied previously but the impact of grape species (under the grape family Vitaceae) was never investigated. Considering the fact, that the phyllosphere microbiome may have profound effects on host plant health and its performance traits, studying the impact of grape species in microbial taxa structuring in the phyllosphere could be of crucial importance. We performed 16S and ITS profiling (for bacteria and fungi respectively) to access genus level characterization of the microflora present in the leaf phyllosphere of five species within this plant family, sampled in two successive years from the repository situated in the Mediterranean. We also performed α and β-diversity analyses with robust statistical estimates to test the impacts of grape species and growing year, over a two-year period. Our results indicated the presence of complex microbial diversity and assemblages in the phyllosphere with a significant effect of both factors (grape species and growing year), the latter effect is being more pronounced. We also compared separate normalization methods for high-throughput microbiome data-sets followed by differential taxa abundance analyses. The results suggested the predominance of a particular normalization method over others. This also indicated the need for more robust normalization methods to study the differential taxa abundance among groups in microbiome research.

The aerial surface of the plant (phyllosphere) is the habitat of complex microbial communities and the structure of this microbiome may be dependent on plant genetic factors, local environment or interactions between them. In this study, we explored the microbial diversity present in the phyllosphere of a very diverse set of grapevine cultivars representing the three genetic pools of the species, grown on an experimental plot at Montpellier (French Mediterranean region). We assessed microbiome variation in the phyllosphere using amplicon sequencing of the 16S rRNA gene and of the internal transcribed spacer (ITS), according to the grapevine genetic pools or cultivars, and organs (i.e. leaves and grape berries). The observed microbiome was complex; out of 542 bacterial genera; Pseudomonas, Pantoea, Sphingomonas, and Acinetobacter were the most abundant and almost ubiquitously present across the samples, and out of 267 fungal genera; Aureobasidium, Alternaria, Mycosphaerella and Aspergillus were most represented. Our results illustrated that the microbial taxa were almost uniformly distributed among the genetic pools and only a few cultivar or genetic pool level differences were found, but a very clear differential taxa abundance was found between the leaf and berry samples. Some genus level associations were also observed with certain genetic pools.

Plant surface or phyllosphere is the habitat of hyperdiverse microbial communities and it is always exposed to the fluctuating environmental factors, which is thought to be one of the potential drivers of microbial community structuring. Impact of grapevine genotypes in variable environmental factors (i.e., at different geographic locations) on the phyllosphere has never been studied and is the main objective of this report. Using high throughput short amplicon sequencing of 16S rRNA genes and internal transcribed spacer (ITS), we analyzed the impacts of genotypes of Vitis Vinifera (coming from three genetic pool), on the microbial (bacterial and fungal) assemblage in the phyllosphere. First, we performed the analysis of the phyllosphere microbiome while using fifteen genotypes that were chosen to maximize intra-specific diversity and grown in two Mediterranean vineyards. Then, the same analysis was performed on five commercially important varieties of Vitis vinifera that were sampled from three different French agro-climatic zones (or terroir: a combination of climate, soils, and human practices). Our study revealed that, at a particular geographic location, genotypes have an impact on microbial assemblage in the phyllosphere and carposphere of leaf and fruit (or berries), respectively, which is more prominent on the carposphere but the effect of terroir was much stronger than the genotype when the leaf phyllosphere of five grapevine varieties grown in different agro-climatic zones was compared. Impacts of the season and exterior plant organs (leaf and berries) on microbial taxa structuring in the phyllosphere was also assessed and presented in this report.

Vitis vinifera subsp. vinifera L., les principales especes de raisins sont cultivees pour la production de fruits et la production de vin dans le monde est un hote naturel d'une grande variete de micro-organismes procaryotes et eucaryotes qui interagissent avec la vigne, ayant des effets benefiques ou phytopathogenes. Ils pourraient egalement jouer un role majeur dans le rendement des fruits, la qualite du raisin, la protection des plantes et, finalement, dans le modele de la fermentation du raisin et la production de vin. La phyllosphere (constituee des parties aeriennes de la plante) est l'un des habitats microbiens les plus repandus sur terre et est un milieu assez neglige, en particulier dans les vignes et de nombreuses questions liees a cet habitat microbien sont toujours sans reponse.Cette these est un effort pour repondre a une question fondamentale en ecologie microbienne: quels sont les facteurs qui determinent le microbiome dans la phyllosphere de la vigne? Les communautes microbiennes de la phyllosphere (PMCs) vivent a l'interface plante-climat et sa capacite a s'etablir, prosperer et se reproduire sur la surface des feuilles ou des fruits depend de plusieurs caracteristiques fonctionnelles microbiennes, comme la capacite de se fixer sur la cuticule et d'utiliser la foliaire. nutriments ainsi que les conditions climatiques dominantes comme la temperature, l'humidite de l'air et la pluie. La chimie des feuilles ou des fruits, la physiologie et la structure morphologique different selon le genotype et l'espece puisque tous ces traits ont une base genetique, et cette variation peut mener a une combinaison differente d'assemblage de PMC parmi les genotypes de plantes. Ainsi, le premier objectif de notre travail etait d'evaluer les impacts des cultivars de vigne (varietes de Vitis vinifera L) et des especes de vigne (especes Vitis entierement differentes) sur l'assemblage du microbiome dans la phyllosphere a un endroit geographique particulier (pour minimiser les effets environnementaux) . Plus tard, les impacts de certains cultivars et terroirs de vigne commercialement importants (representes par trois zones climatiques francaises) ont egalement ete evalues et compares. Les impacts de la saison et des organes exterieurs de la plante (feuilles et baies) sur la structuration des taxons microbiens dans la phyllosphere ont egalement ete evalues et presentes dans ce travail. De plus, des impacts specifiques a l'espece sur le microbiome de la phyllosphere ont egalement ete testes et representes.Dans l'ensemble, notre etude a evalue et compare les nombreuses facettes des facteurs qui peuvent influencer structure du microbiome dans la phyllosphere avec un accent particulier sur la pression de selection relative exercee par le genotype de la vigne et son interaction avec differentes conditions climatiques (ou terroir), ce qui peut ameliorer nos chances de trouver des genes controlant les PMCs sur la phyllosphere. les genes sont reellement importants dans des environnements realistes et probablement ces genes nous donneraient de nouvelles idees pour la selection de nouveaux cepages sains presentant de meilleurs caracteres sur leur phyllosphere. De plus, considerant que les PMC vegetales jouent un role crucial dans la sante et la forme des plantes car elles peuvent moduler la susceptibilite foliaire aux infections, cette etude pourrait egalement etre utile pour developper des methodes de biocontrole innovantes et naturelles ou phytostimulation contre les pathogenes de la vigne. de varietes resistantes innovantes.

AMPK mediates the crosstalk among heteroglycan-induced autophagy and reactive species in RAW 264.7 cells.

Concanavalin A (Con A), a mannose or glucose specific legume lectin, is well known for its anti-proliferative and cytotoxic effect on different types of cancer cells, through its binding to the membrane receptors leading to a major stimulus for the induction of distinct metabolic responses. Recently it has been also been proved that, Con A induces autophagy in hepatoma cells through internalization and mitochondria mediated pathway involving a mitochondrial interacting protein named Bcl2/E1B-19kDa protein-interacting protein 3 (BNIP3). Through this current endeavor, we propose a membrane associated pathway involved in Con A induced autophagy, taking Human cervical cancer (HeLa) cell as a cancer model. Here, we deciphered the role of membrane mediated phosphatidylinositol 3 kinase (PI3K)/Akt/mTOR (mammalian target of rapamycin) and MEK/Extracellular signal-regulated kinases (ERK) pathway in Con A induced autophagy in HeLa cells. Subsequently, we found that Con A treatment suppresses the PI3K/Akt/mTOR and up regulates the MEK/ERK pathway leading to the activation of autophagy. This study will further help us to understand the mechanism behind the autophagic pathway induced by Con A and simultaneously it will strengthen its effective use as a prospective cancer chemo-therapeutic.