TATTOO-seq delineates spatial and cell type-specific regulatory programs in the developing limb

The coordinated differentiation of progenitor cells into specialized cell types and their spatial organization into distinct domains is central to embryogenesis. Here, we developed and applied an unbiased spatially resolved single-cell transcriptomics method to identify the genetic programs underlying the emergence of specialized cell types during mouse limb development and their spatial integration. We identify multiple transcription factors whose expression patterns are predominantly associated with cell type specification or spatial position, suggesting two parallel yet highly interconnected regulatory systems.We demonstrate that the embryonic limb undergoes a complex multiscale reorganization upon perturbation of one of its spatial organizing centers, including the loss of specific cell populations, alterations of preexisting cell states' molecular identities, and changes in their relative spatial distribution. Our study shows how multidimensional single-cell, spatially resolved molecular atlases can allow the deconvolution of spatial identity and cell fate and reveal the interconnected genetic networks that regulate organogenesis and its reorganization upon genetic alterations

CD4+CD25+ regulatory T cells inhibit natural killer cell functions in a transforming growth factor–β–dependent manner

Tumor growth promotes the expansion of CD4+CD25+ regulatory T (T reg) cells that counteract T cell–mediated immune responses. An inverse correlation between natural killer (NK) cell activation and T reg cell expansion in tumor-bearing patients, shown here, prompted us to address the role of T reg cells in controlling innate antitumor immunity. Our experiments indicate that human T reg cells expressed membrane-bound transforming growth factor (TGF)–β, which directly inhibited NK cell effector functions and down-regulated NKG2D receptors on the NK cell surface. Adoptive transfer of wild-type T reg cells but not TGF-β−/− T reg cells into nude mice suppressed NK cell–mediated cytotoxicity, reduced NKG2D receptor expression, and accelerated the growth of tumors that are normally controlled by NK cells. Conversely, the depletion of mouse T reg cells exacerbated NK cell proliferation and cytotoxicity in vivo. Human NK cell–mediated tumor recognition could also be restored by depletion of T reg cells from tumor-infiltrating lymphocytes. These findings support a role for T reg cells in blunting the NK cell arm of the innate immune system

Analysis of Cell Suspensions Isolated from Solid Tissues by Spectral Flow Cytometry

International audienceFlow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to the analysis of a few fluorochromes, currently there are dozens of different fluorescent dyes, and up to 14-18 different dyes can be combined at a time. However, several limitations still impair the analytical capabilities. Because of the multiplicity of fluorescent probes, data analysis has become increasingly complex due to the need of large, multi-parametric compensation matrices. Moreover, mutant mouse models carrying fluorescent proteins to detect and trace specific cell types in different tissues have become available, so the analysis (by flow cytometry) of auto-fluorescent cell suspensions obtained from solid organs is required. Spectral flow cytometry, which distinguishes the shapes of emission spectra along a wide range of continuous wavelengths, addresses some of these problems. The data is analyzed with an algorithm that replaces compensation matrices and treats auto-fluorescence as an independent parameter. Thus, spectral flow cytometry should be capable of discriminating fluorochromes with similar emission peaks and can provide a multi-parametric analysis without compensation requirements.This protocol describes the spectral flow cytometry analysis, allowing for a 21-parameter (19 fluorescent probes) characterization and the management of an auto-fluorescent signal, providing high resolution in minor population detection. The results presented here show that spectral flow cytometry presents advantages in the analysis of cell populations from tissues difficult to characterize in conventional flow cytometry, such as the heart and the intestine. Spectral flow cytometry thus demonstrates the multi-parametric analytical capacity of high-performing conventional flow cytometry without the requirement for compensation and enables auto-fluorescence management

Spectral Cytometry Has Unique Properties Allowing Multicolor Analysis of Cell Suspensions Isolated from Solid Tissues

International audienceFlow cytometry, initially developed to analyze surface protein expression in hematopoietic cells, has increased in analytical complexity and is now widely used to identify cells from different tissues and organisms. As a consequence, data analysis became increasingly difficult due the need of large multi-parametric compensation matrices and to the eventual auto-fluorescence frequently found in cell suspensions obtained from solid organs. In contrast with conventional flow cytometry that detects the emission peak of fluorochromes, spectral flow cytometry distinguishes the shapes of emission spectra along a large range of continuous wave lengths. The data is analyzed with an algorithm that replaces compensation matrices and treats auto-fluorescence as an independent parameter. Thus, spectral flow cytometry should be capable to discriminate fluorochromes with similar emission peaks and provide multi-parametric analysis without compensation requirements. Here we show that spectral flow cytometry achieves a 21-parametric (19 fluorescent probes) characterization and deals with auto-fluorescent cells, providing high resolution of specifically fluorescence-labeled populations. Our results showed that spectral flow cytometry has advantages in the analysis of cell populations of tissues difficult to characterize in conventional flow cytometry, such as heart and intestine. Spectral flow cytometry thus combines the multi-parametric analytical capacity of the highest performing conventional flow cytometry without the requirement for compensation and enabling auto-fluorescence management

Single-Cell Transcriptomic Analysis in the Regenerating Cnidarian Nematostella vectensis

International audienceAbstract Cnidarians have historically served as excellent laboratory models for regenerative development given their capacity to regrow large portions of the adult organism. This capacity is notably absent or poorly developed in the powerful genetic laboratory models Drosophila , C. elegans , and mouse. Increasingly, development of genetic and genomic resources and the application of next-generation sequencing-based techniques in cnidarian systems has further expanded the potential of cnidarian regenerative models. Here, we present a workflow for the characterization of the regenerative response in the sea anemone Nematostella vectensis utilizing fluorescence-activated cell sorting and a plate-based single-cell RNA-sequencing pipeline. This approach can characterize the transcriptional response during regeneration in distinct populations of cells, thus providing a quantitative view of a whole organism process at cellular resolution

Isolation and Characterization of the Immune Cells from Micro-dissected Mouse Choroid Plexuses

International audienceThe brain is no longer considered as an organ functioning in isolation; accumulating evidence suggests that changes in the peripheral immune system can indirectly shape brain function. At the interface between the brain and the systemic circulation, the choroid plexuses (CP), which constitute the blood-cerebrospinal fluid barrier, have been highlighted as a key site of periphery-to-brain communication. CP produce the cerebrospinal fluid, neurotrophic factors, and signaling molecules that can shape brain homeostasis. CP are also an active immunological niche. In contrast to the brain parenchyma, which is populated mainly by microglia under physiological conditions, the heterogeneity of CP immune cells recapitulates the diversity found in other peripheral organs. The CP immune cell diversity and activity change with aging, stress, and disease and modulate the activity of the CP epithelium, thereby indirectly shaping brain function. The goal of this protocol is to isolate murine CP and identify about 90% of the main immune subsets that populate them. This method is a tool to characterize CP immune cells and understand their function in orchestrating periphery-to-brain communication. The proposed protocol may help decipher how CP immune cells indirectly modulate brain function in health and across various disease conditions

Spectral cytometry allows separating fluorescent probes with close emission peaks.

<p>Mouse splenic cells were labeled with fluorescence labeled antibodies. Two different representations of the spectra of dyes with very close emission wavelengths are shown after excitation with the blue: FITC-Ly49D (green) and BB515-CD8 (grey), PerCP-Gr-1 and Cy5.5-PerCP-IgM (red) or the violet laser: V450-IgD (blue) and BV421-CD11c (purple) (<b>A</b>) and can be discriminated on pseudo-color plots after the unmixing algorithm was applied (<b>B</b>).</p

The presence of auto-fluorescent cells does not impair the detection of intra-epithelial lymphocytes, in spectral FCM.

<p>Small intestinal cells comprising epithelial cells and lymphocytes were stained with antibodies recognizing TcRδ-ΠE, TcRβ-Cy7-APC, X∆3-Pacific Blue, Vγ7-APC, CD8-FITC and Vδ4-Cy7-PE. PI was added in the FACS buffer before analysis. The acquisition of the data was done sequentially in the three instruments after appropriate quality control. Doublets were eliminated in the FSC-H/FSC-W. The analysis was done in the Kaluza 1.5 software. Plots show the different steps of the gating strategy. Cells within the FSC-A/SSC-A lymphocyte gate are labeled in blue while cells within the intestinal epithelial cell gate are labeled in red. Arrows point to different population distortions and auto-fluorescence. The first column from the left corresponds to data obtained in instrument B-LSR Fortessa (BD Biosciences); the second, to data obtained in instrument C-Cytoflex (Beckman Coulter); the third and fourth to data obtained in spectral FCM-SP6800 (Sony Inc.) without and with (SP6800 AF) auto-fluorescent management, respectively, after deconvolution.</p

A large subset of auto-fluorescent cells in E17.5 cardiac cell preparations can be included in the fluorescence analysis, in spectral FCM.

<p>Embryonic hearts were isolated and sequentially digested with collagenase with mechanical dissociation at the end of each of seven consecutive digestion cycles. Cell suspensions were stained with antibodies anti TER119 and CD45-PE, Sca-1-Cy5-PE and CD31-APC. Acquisition of the data in the three instruments was done sequentially. Black arrows show auto-fluorescent cells in the two conventional cytometers while these cells are not detected as auto-fluorescent in spectral cytometry and can thus be analyzed for specific fluorescent staining. Instrument A is a Canto II and B a LSR Fortessa (BD Biosciences). B. Auto-fluorescent cells (within the elliptical gate in green) and CD31⁺ cells (rectangular gate in blue), taken as negative control, were sorted into lysis buffer and subjected to Q-RT-PCR that quantified cardiac troponin (<i>TNNT2</i>) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159961#pone.0159961.ref013" target="_blank">13</a>] and atrial light chain-2 (<i>MYL7</i>) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159961#pone.0159961.ref014" target="_blank">14</a>] transcripts specific for cardiomyocytes and not found in endothelial, stromal or hematopoietic cells. a.u. arbitrary units calculated relative to the expression of the house-keeping transcript GAPDH.</p

An 18-color antibody panel for the analysis of murine spleen cells.

<p>Multi-color antibodies panel with 18 different antibody-labeled fluorochromes, excited by the 488nm laser (blue) and by the 405nm laser (purple) (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159961#pone.0159961.g005" target="_blank">Fig 5</a>). The complex emission spectra of all fluorochromes are shown in the 488nm (upper left panel) and in the 405nm lasers (upper right panel) and in reference fluorochrome spectra shape with each colored curve corresponding to a different fluorochrome (lower panel)(<b>B</b>). <b>C</b>. The contour plots show the discriminative capacity of this multi-parametric analysis to separate the different populations of B, T, NK, dendritic or myeloid cells. The brown arrow shows rare subsets of NK and myeloid cells. The analysis was done in the FlowJo software after deconvolution.</p