Development, application and computational analysis of high-dimensional fluorescent antibody panels for single-cell flow cytometry

The interrogation of single cells is revolutionizing biology, especially our understanding of the immune system. Flow cytometry is still one of the most versatile and high-throughput approaches for single-cell analysis, and its capability has been recently extended to detect up to 28 colors, thus approaching the utility of cytometry by time of flight (CyTOF). However, flow cytometry suffers from autofluorescence and spreading error (SE) generated by errors in the measurement of photons mainly at red and far-red wavelengths, which limit barcoding and the detection of dim markers. Consequently, development of 28-color fluorescent antibody panels for flow cytometry is laborious and time consuming. Here, we describe the steps that are required to successfully achieve 28-color measurement capability. To do this, we provide a reference map of the fluorescence spreading errors in the 28-color space to simplify panel design and predict the success of fluorescent antibody combinations. Finally, we provide detailed instructions for the computational analysis of such complex data by existing, popular algorithms (PhenoGraph and FlowSOM). We exemplify our approach by designing a high-dimensional panel to characterize the immune system, but we anticipate that our approach can be used to design any high-dimensional flow cytometry panel of choice. The full protocol takes a few days to complete, depending on the time spent on panel design and data analysis

Aptamers against mouse and human tumor-infiltrating myeloid cells as reagents for targeted chemotherapy

Local delivery of anticancer agents has the potential to maximize treatment efficacy and minimize the acute and long-term systemic toxicities. Here, we used unsupervised systematic evolution of ligands by exponential enrichment to identify four RNA aptamers that specifically recognized mouse and human myeloid cells infiltrating tumors but not their peripheral or circulating counterparts in multiple mouse models and from patients with head and neck squamous cell carcinoma (HNSCC). The use of these aptamers conjugated to doxorubicin enhanced the accumulation and bystander release of the chemotherapeutic drug in both primary and metastatic tumor sites in breast and fibrosarcoma mouse models. In the 4T1 mammary carcinoma model, these doxorubicin-conjugated aptamers outperformed Doxil, the first clinically approved highly optimized nanoparticle for targeted chemotherapy, promoting tumor regression after just three administrations with no detected changes in weight loss or blood chemistry. These RNA aptamers recognized tumor infiltrating myeloid cells in a variety of mouse tumors in vivo and from human HNSCC ex vivo. This work suggests the use of RNA aptamers for the detection of myeloid-derived suppressor cells in humans and for a targeted delivery of chemotherapy to the tumor microenvironment in multiple malignancies

Indoleamine 2,3-dioxygenase is a signaling protein in long-term tolerance by dendritic cells

Regulation of tryptophan metabolism by indoleamine 2,3-dioxygenase (IDO) in dendritic cells (DCs) is a highly versatile modulator of immunity. In inflammation, interferon-\u3b3 is the main inducer of IDO for the prevention of hyperinflammatory responses, yet IDO is also responsible for self-tolerance effects in the longer term. Here we show that treatment of mouse plasmacytoid DCs (pDCs) with transforming growth factor-\u3b2 (TGF-\u3b2) conferred regulatory effects on IDO that were mechanistically separable from its enzymic activity. We found that IDO was involved in intracellular signaling events responsible for the self-amplification and maintenance of a stably regulatory phenotype in pDCs. Thus, IDO has a tonic, nonenzymic function that contributes to TGF-\u3b2-driven tolerance in noninflammatory context

Most representative gene sets associated with the <i>Inflammation</i>, <i>Early Anti-Inflammation</i> and <i>Anti-Inflammation</i> functional groups.

<p>KEGG, Biocarta, and Reactome gene sets have been obtained from the C2: <i>curated gene sets collection</i> of the Molecular Signatures Database. Gene sets were defined as significantly enriched if FDR<0.05 when using Pearson as metric and 1,000 permutations of gene sets. The complete list of the Gene sets identified by GSEA is available with the authors.</p

Gene expression validation by qPCR.

<p>Fold-expression levels determined by qPCR for the 10 genes selected in the <i>Inflammation</i> (<i>PPARG</i>, <i>IL6</i>, <i>TNFA</i>, <i>IL7R</i>), <i>Early Anti-Inflammation</i> (<i>CD163</i>), <i>Anti-Inflammation</i> (<i>CEBPA</i>), <i>Inflammation Driven Differentiation</i> (<i>PPARD</i>), <i>Positive Differentiation</i> (<i>MMP9</i> and <i>MAFB</i>), and <i>Negative Differentiation</i> (<i>KLF4</i>) groups. The mean expression values ± SEM from six different donors are reported. Statistical significance was calculated with ANOVA followed by Fisher's test for significant differences between two consecutive experimental time points. * <i>P</i><.05; ** <i>P</i><.001; *** <i>P</i><.0001.</p

Correlation between M1/M2 polarization and functional groups.

<p>Association of gees that are up- or downregulated in M1 and M2 cells polarisation (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087680#pone-0087680-g003" target="_blank">Figure 3A and B</a>) with the functional groups defined from the analysis of the <i>in vitro</i> model of inflammation (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087680#pone-0087680-g002" target="_blank">Figure 2</a>).</p

Graphic representation of the kinetic development of inflammation in the human monocyte-based <i>in vitro</i> model.

<p>Freshly isolated human blood monocytes were first exposed to the chemokine CCL2 for 2°C, then to LPS (from 2 h), TNF-α (from 3 h), and IFN-γ (from 7 h) at 39°C. At 14 h the inflammatory stimuli were washed off, the temperature brought back to 37°C, and fresh medium containing IL-10 added. At 24 h monocytes were exposed to TGF-β until the end of the experiment.</p

Differentially expressed genes in M1 and M2 macrophages <i>vs.</i> monocytes.

<p>Heat-maps representing the fold-expression levels of gene lists identified by SAM as statistically downregulated (green) or upregulated (red) in M1 and M2 samples compared to fresh unstimulated monocytes. The lists excluded genes that are modulated in both M1 and M2 <i>vs.</i> monocytes. (A) Fold-expression levels in monocytes and M1 macrophages of the meta-database for the 98 genes associated to monocyte-to-M1 differentiation. (B) Fold-expression levels in monocytes and M2 macrophages of the meta-database for the 107 genes associated to monocyte-to-M2 differentiation. (C) Fold-expression levels of the 98 monocyte-to-M1 genes assessed in the 60 samples of our <i>in vitro</i> model of inflammation. (D) Fold-expression levels of the 107 monocyte-to-M2 genes assessed in the 60 samples of our <i>in vitro</i> model of inflammation.</p

The reform of law studies

Artykuł prezentuje argumenty w toczącej się dziś dyskusji nad programem studiów prawniczych. Spierają się zwolennicy utrzymania istniejącej grupy przedmiotów ogólnych i teoretycznych. Ich przeciwnicy opowiadają się za zwiększeniem praktycznego charakteru studiów i zmniejszenia listy przedmiotów ogólnych.***This paper presents arguments raised in the current discussion in Poland on the syllabus of reformed law studies. The dispute concerns the proportion between general and theoretical subjects and the recently advocated increased number of subjects teaching practical skills at the cost of lesser academic content