Quantification of biotic stresses on aerial parts of plants using Chlorophyll Fluorescence Imaging and Image Analysis

Image analysis is increasingly used in plant phenotyping. Among the various imaging techniques available for plant phenotyping, Chlorophyll Fluorescence Imaging is particularly su itable for the imaging of biotic and abiotic stresses on the aerial parts of plants. Numerous chlorophyll fluorescence parameters may be measured or calculated, but only some of them may provide useful contrasts for the quantification of a given stress on leaves. In the perspective of high throughput phenotyping of biotic stresses on plants, we developed automated procedures to identify Chlorophyll Fluorescence parameters of interest for the quantification of a given biotic stress on large image datasets. The outputs of the automated procedures enable: - the visualization of the whole dataset, by providing contact sheets for each of the chlorophyll fluorescence parameter tested. - the visualization of basic statistics : radial - plot, box - plot and Mann - Whitney tests based on the mean intensity of each parameters are provided to compare the various treatments performed. - images are clustered based on histograms associated to each images, thereby enabling the clustering of images leaves displaying leaves of si milar phytosanitary status. - the objective quantification of disease incidence on each leaf tested. The quantification can discriminate varois stages of symptom development such as necrotic tissues, wilted and chlorotic tissues, and impacted tissues that do not display any symptoms visible to the eye.

A size and space structured model of tumor growth describes a key role for protumor immune cells in breaking equilibrium states in tumorigenesis

International audienceSwitching from the healthy stage to the uncontrolled development of tumors relies on complicated mechanisms and the activation of antagonistic immune responses, that can ultimately favor the tumor growth. We introduce here a mathematical model intended to describe the interactions between the immune system and tumors. The model is based on partial differential equations, describing the displacement of immune cells subjected to both diffusion and chemotactic mechanisms, the strength of which is driven by the development of the tumors. The model takes into account the dual nature of the immune response, with the activation of both antitumor and protumor mechanisms. The competition between these antagonistic effects leads to either equilibrium or escape phases, which reproduces features of tumor development observed in experimental and clinical settings. Next, we consider on numerical grounds the efficacy of treatments: the numerical study brings out interesting hints on immunotherapy strategies, concerning the role of the administered dose, the role of the administration time and the interest in combining treatments acting on different aspects of the immune response. Such mathematical model can shed light on the conditions where the tumor can be maintained in a viable state and also provide useful hints for personalized, efficient, therapeutic strategies, boosting the antitumor immune response, and reducing the protumor actions.Le passage du stade sain à la croissance incontrôlée des tumeurs repose sur des mécanismes compliqués et l'activation de réponses immunitaires antagonistes, qui peuvent à terme favoriser la croissance tumorale. Nous introduisons ici un modèle mathématique destiné à décrire les interactions entre le système immunitaire et les tumeurs. Le modèle est basé sur des équations aux dérivées partielles, décrivant le déplacement de cellules immunitaires soumises à la fois à des mécanismes de diffusion et de chimiotactisme, dont la force est déterminée par le développement des tumeurs. Le modèle prend en compte la double nature de la réponse immunitaire, avec l'activation des mécanismes antitumoraux et protumoraux. La compétition entre ces effets antagonistes conduit soit à des phases d'équilibre, soit à des phases d'échappement, qui reproduisent les caractéristiques du développement tumoral observé dans des contextes expérimentaux et cliniques. Ensuite, nous considérons sur des bases numériques l'efficacité des traitements : l'étude numérique apporte des pistes intéressantes sur les stratégies d'immunothérapie, concernant le rôle de la dose administrée, le rôle du temps d'administration et l'intérêt de combiner des traitements agissant sur différents aspects du système immunitaire. Un tel modèle mathématique peut faire la lumière sur les conditions dans lesquelles la tumeur peut être maintenue dans un état viable et également fournir des pistes utiles pour des stratégies thérapeutiques personnalisées et efficaces, renforçant la réponse immunitaire antitumorale et réduisant les actions protumorales

NKp46+ Innate Lymphoid Cells Dampen Vaginal CD8 T Cell Responses following Local Immunization with a Cholera Toxin-Based Vaccine

International audienceInnate and adaptive immune cells work in concert to generate efficient protection at mucosal surface. Vaginal mucosa is an epithelial tissue that contains innate and adaptive immune effector cells. Our previous studies demonstrated that vaginal administration of Cholera toxin -based vaccines generate antigen-specific CD8 T cells through the stimulation of local dendritic cells (DC). Innate lymphoid cells (ILC) are a group of lymphocytes localized in epithelial tissues that have important immune functions against pathogens and in tissue homeostasis. Their contribution to vaccine-induced mucosal T cell responses is an important issue for the design of protective vaccines. We report here that the vaginal mucosa contains a heterogeneous population of NKp46+ ILC that includes conventional NK cells and ILC1-like cells. We show that vaginal NKp46+ ILC dampen vaccine-induced CD8 T cell responses generated after local immunization. Indeed, in vivo depletion of NKp46+ ILC with anti-NK1.1 antibody or NKG2D blockade increases the magnitude of vaginal OVA-specific CD8 T cells. Furthermore, such treatments also increase the number of DC in the vagina. NKG2D ligands being expressed by vaginal DC but not by CD8 T cells, these results support that NKp46+ ILC limit mucosal CD8 T cell responses indirectly through the NKG2D-dependent elimination of vaginal DC. Our data reveal an unappreciated role of NKp46+ ILC in the regulation of mucosal CD8 T cell responses

Tumor-Associated Neutrophils Dampen Adaptive Immunity and Promote Cutaneous Squamous Cell Carcinoma Development

Cutaneous squamous cell carcinoma (cSCC) development has been linked to immune dysfunctions but the mechanisms are still unclear. Here, we report a progressive infiltration of tumor-associated neutrophils (TANs) in precancerous and established cSCC lesions from chemically induced skin carcinogenesis. Comparative in-depth gene expression analyses identified a predominant protumor gene expression signature of TANs in lesions compared to their respective surrounding skin. In addition, in vivo depletion of neutrophils delayed tumor growth and significantly increased the frequency of proliferating IFN-γ (interferon-γ)-producing CD8+ T cells. Mechanisms that limited antitumor responses involved high arginase activity, production of reactive oxygen species (ROS) and nitrite (NO), and the expression of programmed death-ligand 1 (PD-L1) on TAN, concomitantly with an induction of PD-1 on CD8+ T cells, which correlated with tumor size. Our data highlight the relevance of targeting neutrophils and PD-L1-PD-1 (programmed death-1) interaction in the treatment of cSCC

Functional properties of vaginal Eomes⁺ NK cells and Eomes^- ILC1-like cells.

<p>(A) Bidimensional dot plots (left panel) show expression of CD27 and CD11b on gated Eomes⁺ NKp46⁺ CD3^- cells from spleen, GLN and vaginas of naive C57BL/6 mice. Numbers in quadrants represent the percentage of cells for each subset. Histograms bars (right panel) represent results from five independent experiments expressed as mean values + SEM, <i>n</i> = 10 mice. <i>**p<0</i>.<i>01</i>, <i>***p<0</i>.<i>001</i>, <i>****p<0</i>.<i>0001</i> Mann-Whitney <i>U</i> test. Spl.: spleen; GLN: genital lymph nodes; Vag.: vagina. Frequencies of CD107a⁺ and IFN-γ⁺ producing Eomes⁺ NK cells (B) and Eomes^- ILC1-like cells (C) in cell suspensions from spleen, GLN and vagina of naive C57BL/6 mice. Cells (0.5 million/well) were stimulated <i>in vitro</i> for 4 hours with plate-bound isotype control (IC), anti NK1.1 mAb, PMA/ionomycin, IL-12 + IL-18 cytokines or YAC-1 tumor cells. CD107a and IFN-γ expression on cells were assessed by flow cytometry. Histogram bars represent results from three independent experiments expressed as mean percentages + SEM, <i>n</i> = 5 mice. <i>**p<0</i>.<i>01</i>, <i>***p<0</i>.<i>001</i>, Mann-Whitney <i>U</i> test. (D) Bidimensional dot plots (left panel) show expression of TNFα and TRAIL on gated Eomes^- ILC1 and Eomes⁺ NK cells from liver and vaginas of naive C57BL/6 mice after stimulation <i>in vitro</i> for 4 hours with PMA/ionomycin. Numbers in quadrants represent the percentage of cells for each subset. Results are representative of two independent experiments with n = 4 mice.</p

Dynamics of CD11c+ dendritic cell subsets in lymph nodes draining the site of intestinal nematode infection

Helminth parasites drive dominant Th2 responses through an as yet unidentified pathway. We have previously shown that the rodent gastrointestinal nematode Nippostrongylus brasiliensis secretes products which selectively activate in vitro-derived dendritic cells to induce Th2 responses on in vivo transfer. We now show that, during active infection with this parasite, the draining mesenteric lymph node dendritic cell population is altered significantly. Although there is substantial expansion of DC numbers during infection, the CD86hi-CD8αint-CD11b− subset is markedly diminished, and expression levels of CD40, CD86 and CD103 are reduced. Notably, the reduced frequency of CD8αint DCs is evident only in those mesenteric lymph nodes draining the anterior site of infestation. In infections with the longer lived Heligmosomoides polygyrus, the proportion of CD8αint DCs in the MLNC falls to below 10% of total DC numbers by 35 days post-infection. Further, infection alters TLR responsiveness, as IL-12 production (as measured by ex vivo intracellular staining of CD11c+ DCs) in response to LPS stimulation is reduced, while IL-6, TNF-α and in particular, IL-10 all increase following infection with either nematode parasite. These changes suggest the possibility that helminth parasites modulate gastrointestinal immunity both by inhibiting migration of CD8αint DCs to the draining lymph nodes, and modifying DC responsiveness in a manner which favours a Th2 outcome

Identification of vaginal NKp46⁺ CD3^- cells.

<p>(A) Frequencies (left panel) and numbers (right panel) of NKp46⁺ CD3^- cells among CD45⁺ leukocytes from spleen, genital lymph nodes (GLN) and vagina of naive C57BL/6 mice. Histogram plots represent results from five independent experiments and are expressed as mean values + SEM, <i>n</i> = 10 mice. (B) Immunofluorescence staining of frozen sections from mouse vagina and spleen stained with anti-CD3 (green) and anti-NKp46 (red) antibodies. Nuclei were visualized with DAPI (blue). White arrows indicate NKp46⁺ CD3^- cells; E: epithelium; LP: lamina propria. WP: white pulp; RP: red pulp. White dotted lines delineate the epithelium from the lamina propria in the vagina and the white pulp from the red pulp in the spleen. Original magnification: x40 (vagina) and x20 (spleen). Data are representative of three independent experiments.</p